DEVELOPMENTAL DYNAMICS 211:1–10 (1998) Developmental Expression and Localization of the Catalytic Subunit of Protein Phosphatase 2A in Rat Lung CHUN XUE,1 FELICE HELLER,2 ROGER A. JOHNS,1 AND ALLEN D. EVERETT2* 1Department of Anesthesiology, University of Virginia Health Sciences Center, Charlottesville, Virginia 2Department of Pediatrics, University of Virginia Health Sciences Center, Charlottesville, Virginia ABSTRACT Protein phosphatase type-2A (PP2A) is a highly conserved serine/threonine phosphatase known to play a key role in cell proliferation and differentiation in vitro, but the role of PP2A in mammalian embryogenesis remains unexplored. No particular information exists as to the tissue or cell specific expression of PP2A or the relevance of PP2A expression to mammalian development in vivo. To examine expression of PP2A during mammalian lung development, we studied fetal rats from day 14 of gestation (the lung bud is formed on day 12 of gestation) to parturition. Western analysis with a specific PP2A catalytic subunit antibody identified a single 36 kDa protein, with protein levels two-fold higher in the 17 and 19 day embryonic lung as compared to the adult. With in situ hybridization and immunohistochemistry, both mRNA and protein for PP2A were localized equally to the epithelial lining of the embryonic lung airway and the surrounding mesenchyme in the 14 day embryonic lung. With maturation of the lung, PP2A becomes highly expressed in respiratory epithelium. The highest level of expression was in the earliest developing airways with columnar epithelium (the pseudoglandular stage, 15–18 days of gestation). There was a decrease in expression with the transformation to cuboidal epithelium by day 20 of gestation. This was most noticeable in the developing bronchial epithelium of the 19 and 20 day gestation lungs where only an occasional cell continues to express PP2A. Mesenchymal hybridization was most obvious in early endothelial cells of forming vascular channels at 17–19 days of gestation. PP2A respiratory epithelial expression mimicked the centrifugal development of the respiratory tree where the highest expression was in the peripheral columnar epithelium (15–18 days gestation) with only an occasional central bronchiolar cell continuing to express PP2A at 19 and 20 days gestation. Endothelial hybridization decreased with muscularization of large pulmonary arteries with low levels of expression detected in bronchial or vascular smooth muscle. In the newborn lung PP2A expression was decreased, but detectable in alveolar epithelium and vascular endothelium. In summary; 1) PP2A mRNA and protein exhibit cell r 1998 WILEY-LISS, INC. specific expression during rat lung development; 2) PP2A is highly expressed in the respiratory epithelium of the fetal rat lung and is temporally related to the maturation of the bronchial epithelium; 3) and the PP2A subunit is highly expressed in early vascular endothelium, but not smooth muscle of the rat lung. Dev. Dyn. 1998;211:1–10. r 1998 Wiley-Liss, Inc. Key words: epithelium; lung development; fetus; serine/threonine phosphatase; endothelium; immunohistochemistry; in situ hybridization INTRODUCTION Epithelial and mesenchymal interactions are necessary for the development of many organ systems including those of the gastrointestinal, integument, urogenital, and respiratory systems. Lung development serves as a classical model for such biologic interactions (Minoo et al., 1994). Formed initially as an outpouching of the primitive foregut, the lung subsequently undergoes growth and branching of the primitive respiratory epithelium into the surrounding mesenchyme to form the bronchial tree. A complex process of interactions among cells, cytokines, extracellular matrix, and cell membrane receptors is necessary for lung morphogenesis and regional specification of the respiratory system. Although there must be some mechanism to link extracellular signals to intracellular reactions such as gene expression (signal transduction), in the developing lung, neither the components nor their functions are well understood. An essential mechanism in the regulation of signal transduction involves the activities of phosphoproteins capable of reversible phosphorylation and dephosphorylation (Cohen et al., 1989; Mumby et al., 1993; Hemmings et al., 1994). Protein phosphatases exist in eukaryotic cells to regulate the phosphoryla- Grant sponsor: March of Dimes Basil O’Connor Fellowship Award; Grant sponsor: National Institutes of Health; Grant numbers: K08HL02937-02, R01-HL39706, and R01-GM49111; Grant sponsor: American Heart Associate–Virginia Affiliate. Dr. Xue is currently at the Department of Asthma and Allergy Research, Pharmaceuticals Division, Novartis, CH 4002 Basel, Switzerland. *Correspondence to: Dr. Allen D. Everett, M.D., University of Virginia Health Sciences Center, MR4 Building, Box 14, Charlottesville, VA 22908. Received 4 June 1997; Accepted 11 September 1997 2 XUE ET AL. tion state of phosphoproteins, and are largely divided into protein tyrosine and serine/threonine phosphatases as determined by their amino acid substrates. Of the serine/threonine protein phosphatases, protein phosphatase type-2A (PP2A) is highly conserved in all eukaryotic cells and accounts for a large portion of total cellular phosphatase activity (Cohen et al., 1989; Hemmings et al., 1994). PP2A is essential for a variety of cellular functions including signal transduction, cell cycle regulation, cell transformation, and cell fate determination (Hemmings et al., 1994). PP2A is a trimeric holoenzyme, composed of a core dimer plus a third subunit. The holoenzyme consists of a 36 kDa catalytic (C) subunit (Stone et al., 1987; Khew-Goodall et al., 1991), a 63 kDa structural (A) subunit (Hemmings et al., 1990), and a third variable, regulatory (B) subunit (Hemmings et al., 1994; Tehrani et al., 1996; McCright et al., 1996). The B subunit is variable, ranging from 54 to 130 kDa and serves to confer distinct properties on the enzyme for substrate specificity (Hemmings et al., 1994; Kamibayaashi et al., 1992; Cegielska et al., 1994). The catalytic subunit of PP2A is highly conserved (Hemmings et al., 1994), the human structure having 70% homology to yeast Saccharomyces cerevisiae (Arndt et al., 1989) and 95% identity with Drosophila (Mayer-Jaekel et al., 1992, 1993). There is no evidence that the free catalytic subunit exists in cells (Mumby et al., 1993). Although the in vitro biochemical aspects of PP2A are well known, the role of PP2A in mammalian development is largely unexplored. For PP2A to play a role in lung development it would be expected that there be developmental differences in PP2A subunit expression and activity. As shown by Warburton and Cohen (1988), PP2A activity is developmentally upregulated in the rat lung. To date no information exists on the developmental, cell specific expression of PP2A in any organ, including the lung. To begin to explore a potential role for PP2A in lung development, the present study determined the cellular expression pattern of the 36 kDa catalytic subunit of PP2A (PP2A) in development of the rat fetal lung. As the catalytic subunit does not exist alone in nature but always as an active enzyme coupled at least to the A structural subunit (Mumby et al., 1993) we used PP2A as a marker for expression of the PP2A holoenzyme in the developing lung using Western analysis, in situ hybridization, and immunohistochemistry. RESULTS Western Analysis of PP2A Western analysis of whole lung homogenates for PP2A protein was performed as shown by the representative blot in Figure 1A. Using a specific polyclonal peptide antibody against the carboxy terminal portion of PP2A a single 36 kDa protein was detected that co-migrated with the PP2A control protein (not shown) as previously reported for PP2A (Stone et al., 1987; Khew-Goodall et al., 1991; Martin et al., 1994). As A Fig. 1. Representative Western analysis of PP2A protein levels in the developing lung. A: Total lung protein (50 µg) from 14 day (E14, n 5 29), 17 day (E17, n 5 27), 19 day (E19, n 5 18), and 20 day (E20, n 5 17) gestation, newborn (NB, n 5 9) and adult (AD, n 5 2) rats was analyzed by SDS-PAGE with Western blotting for PP2A protein levels. Protein standard markers are shown on left. B: Densitometric analysis of PP2A protein levels is shown. shown (Fig. 1A,B), PP2A protein is abundant in the developing lung with protein levels two-fold higher in the 17 and 19 day embryonic lung as compared to the adult. Therefore PP2A protein demonstrates significant developmental regulation in the lung with protein levels highest at the time of maximal growth of the lung. In Situ Hybridization and Immunohistochemistry 14 day gestation lung. To map the cell specific mRNA and protein expression of PP2A in the developing lung, in situ hybridization and immunohistochemical methods were utilized. PP2A mRNA was readily detected in the developing lung at 14 day of gestation (Fig. 2, the lung bud is formed during day 12 of gestation). PP2A mRNA (Fig. 2A,B) and protein (Fig. 4A) were widely expressed in the epithelium and mesenchyme of the developing lung bud. 17 day gestation lung. By 17 days of gestation (Fig. 2C–G) the lung airway has undergone extensive branch- PROTEIN PHOSPHATASE 2A EXPRESSION IN DEVELOPING LUNG Fig. 2. PP2A mRNA localization in the 14 and 17 day gestation lung. In situ hybridization for PP2A in 14 day gestation rat lung is shown in A and B and 17 day lung in C–G. The anti-sense cRNA probe was used in A, C, E–G, and the control sense probe in B and D. A,B: The respiratory epithelium in the 14 day lung bud is identified by arrows. C–G: Hybridization to small developing airways (small arrows), larger bronchioles (large 3 arrows), endothelial cells (arrow heads), and the pulmonary artery (long arrow) is shown. F is a magnification of E and demonstrates hybridization to endothelial cells of developing vascular channels (arrow heads). G demonstrates high expression of PP2A in the respiratory epithelium of immature forming airways (arrows) and decreased expression in epithelium lining bronchioles (B). 4 XUE ET AL. ing. At this stage, PP2A mRNA hybridization was increased relative to the 14 day lung (Fig. 2A) and continues to map predominately to the now glandular airway epithelium (Fig. 2C–G). A phenotypic gradient of PP2A mRNA expression was demonstrated between the more mature central bronchiolar and peripheral (least mature) respiratory epithelial cells. As shown in Figure 2E–G PP2A is highly expressed in the respiratory epithelium of the least mature peripheral airway epithelium. Whereas, expression is markedly decreased in the more mature bronchiolar (‘‘B’’ in panel G and large arrow panel E, Fig. 2) epithelial cells. Mesenchymal expression of PP2A in the 17 day lung localized predominately to endothelial cells of forming vascular channels (Fig. 2F) identified by the presence of red blood cells. The specificity of the in situ hybridization is shown in the control section incubated with the digoxigenin labeled mRNA probe (Fig. 2D). As shown, there is an absence of nonspecific hybridization in the fetal lung demonstrating the specificity of the cRNA probe. PP2A mRNA expressing cells in the 17 day gestation lung were also seen to contain PP2A protein (Fig. 4B). Immunocytochemistry with a PP2A specific antibody (Fig. 4B) demonstrated that respiratory epithelial cells as well as the endothelial cells of forming vascular channels were all immunopositive for PP2A (Fig. 4B). 19 day gestation lung. At 19 days of gestation, as the airway branches extensively, PP2A mRNA continues to be expressed at a very high level in the airway epithelium (Fig. 3A,C–E). However, the pattern of expression is higher within the epithelium of small canalicular structures in the peripheral and less mature portions of the lung (small arrows in Fig. 3D,E) rather than in the large, central more mature airways (large arrows in Fig. 3D,E). The quantity of PP2A mRNA hybridization clearly decreased in the transition from a more cuboidal to flattened epithelial cell type in the more mature airways (Fig. 3A,C–E). In particular the bronchiolar epithelial cells (‘‘B’’ in Fig. 3A,C,D) showed very little PP2A mRNA hybridization relative to the more glandular respiratory epithelial cells. There did appear to be a gradient of expression in the bronchiolar epithelium with higher mRNA expression in the peripheral than in the central bronchiolar epithelium (Fig. 3C). Expression in the mainstem bronchus epithelium was higher in cells nearest the lumenal surface of the bronchus (Fig. 3D). Intense hybridization to endothelial cells lining forming vascular channels (Fig. 3E arrow heads) was seen. However endothelial and smooth muscle cells of mature muscular pulmonary arteries (Fig. 3A arrows and D ‘‘PA’’) expressed PP2A at background levels. The specificity of the in situ hybridization is shown in Figure 3B using the control sense probe. As shown incubation with the control sense probe demonstrated a complete lack of hybridization to the airway epithelium and lung parenchyma. Immunocytochemically PP2A continued to map to endothelial cells of forming vascular channels (Fig. 4C arrow heads) and airway epithelium (Fig. 4C,D, small arrows). The PP2A protein was expressed in both cytoplasmic and nuclear compartments of endothelial (Fig. 4C, arrowheads) and respiratory epithelial cells (Fig. 4C,D, small arrows). The nuclear localization of PP2A protein in respiratory epithelial cells is shown in Figure 4D (small arrows) and decreases in cells of larger airways (Fig. 4D, large arrow). Likewise endothelial cells of more mature blood vessels (Fig. 4D, long arrow) expressed less PP2A protein. Newborn lung. After birth, alveolar cells continued to express PP2A mRNA (Fig. 5A, arrows) whereas most of the bronchial epithelial cells in the aerated lung were negative for PP2A mRNA (Fig. 5C, arrows). Endothelial cells lining capillaries and small peripheral arteries (Fig. 5A,E, arrowheads) continued to express PP2A mRNA at high levels however expression in large muscular arteries was less obvious (Fig. 5F, arrowheads). Hybridization to the sense control probe is shown (Fig. 5B,D) and demonstrates the specificity of the results. PP2A protein expression was decreased as compared to the 19 day gestation lung but continued to be detectable in alveolar epithelial and endothelial cells (Fig. 4E). No immunostaining was detected in a serial section using nonspecific rabbit IgG as a control (Fig. 4F). DISCUSSION Considering the conserved nature of PP2A and the abundant number of regulatory units available Mumby and Walter (1993) have proposed that the catalytic subunit would not show cell and tissue specific regulation. However, the present study shows regulated expression at high levels of PP2A mRNA and protein in the developing bronchiolar epithelium and endothelial cells. In addition western analysis demonstrated a two fold decrease in PP2A protein from 19 day gestation to the adult. Thus paralleling the maturation of the lung airway. Similarly, endothelial cells of the pulmonary blood vessels demonstrate increased expression of PP2A protein and mRNA in the fetus as compared to the newborn. Therefore, PP2A demonstrates cell specific regulation in expression during development, an unexpected result based on existing knowledge. Developmental regulation of PP2A mRNA has been demonstrated previously by Northern analysis in whole Drosophila (Mayer-Jaekel et al., 1993) and Xenopus (Van Hoof et al., 1995) embryos although cell and tissue specific expression of PP2A was not demonstrated. This cell specific and maturational associated localization of PP2A to developing lung epithelium suggests a role for PP2A in the epithelial-mesenchymal interactions necessary for normal lung development. Embryologically the lung is of endodermal origin forming as an outpouching of the primitive gut into the surrounding mesenchyme. Although a great number of growth factors and their respective receptors have been identified that affect lung development (Minoo et al., 1994) the complex cascade of down stream events mediating their effect are largely unknown. The complex process of lung PROTEIN PHOSPHATASE 2A EXPRESSION IN DEVELOPING LUNG Fig. 3. PP2A mRNA localization in the 19 day gestation lung. In situ hybridization for PP2A in 19 day gestation rat lung is shown with the antisense probe in A, C–E, and the sense control probe in B. Hybridization to bronchiolar epithelium (B), larger more mature airways (large arrows), small forming airways (small arrows), pulmonary arteries (long arrows and PA), and endothelium of small forming vascular channels 5 (arrow heads) is shown in A–E. D demonstrates the minimal hybridization to the vascular smooth muscle and endothelium of the pulmonary artery (PA). E demonstrates the high level of expression of PP2A in the small forming airways (small arrows) with decreased expression in larger airways (large arrows) and endothelium of forming vascular channels (arrow head). Fig. 4. Mapping PP2A protein expression in the 14, 17, 19 day gestation and newborn lung. PP2A protein was detected by immunocytochemistry in the 14 day (A), 17 day (B), 19 day (C and D) gestation, and newborn (E) rat lung. F is a slide of newborn lung incubated with nonspecific IgG as a control. Arrows in A indicate staining of the respiratory epithelium in the 15 day lung bud. Staining of the respiratory epithelium of small forming airways (short arrows), endothelium of forming vascular channels (arrow heads), larger more mature airways (large arrows) and arteries (long arrows) is shown in B–F. D demonstrates PP2A protein localized to the nucleus of epithelial cells lining developing airways (short arrows). In D, expression of PP2A protein is decreased in the endothelial of larger blood vessels (long arrow) and the epithelium of larger airways (large arrows). Staining of alveolar cells (arrows) and capillary endothelium (arrow head) in the newborn lung is shown in E. PROTEIN PHOSPHATASE 2A EXPRESSION IN DEVELOPING LUNG 7 Fig. 5. PP2A mRNA expression in the newborn lung. In situ hybridization with a anti-sense probe (A,C,E, and F) and control sense probe (B and D) is shown. In A hybridization to alveolar cells (arrows) and capillary endothelial cells (arrowhead) was detected. A serial section hybridized with the sense control probe (B) demonstrated no background hybridiza- tion. Hybridization to bronchial epithelium was very low as shown by the arrows in the antisense probe in C and control sense probe in D. Hybridization was still detectable in endothelium of small muscular arteries in the periphery of the lung (E, arrowheads) whereas little hybridization was detected in the endothelium (F, arrowhead) or smooth muscle of larger arteries (F). morphogenesis requires the coordinate signal transduction of information from the plasma membrane to the cell interior. Reversible phosphorylation and dephos- phorylation of proteins is an essential mechanism regulating the activities of proteins involved in cell signaling (Mumby et al., 1993). PP2A is a significant 8 XUE ET AL. down stream signaling molecule playing a role in cell fate determination (Uemura et al., 1993) and serves as a negative regulator of progression through the cell cycle (Clarke et al., 1993) and growth and proliferation (Sontag et al., 1993). A plausible explanation is that increased PP2A expression is related to PP2A regulation of gene transcription (Alberts et al., 1993). Increased PP2A at the time of branching morphogenesis and the decline in PP2A at its completion suggests that PP2A is regulating expression of genes possibly necessary for the differentiation of the respiratory epithelial cells. This theory is supported by the identification of nuclear and cytoplasmic localization of PP2A protein in endothelium and respiratory epithelial cells in this study and the recent identification of specific B regulatory subunits that target PP2A to the nucleus (Tehrani et al., 1996; McCright et al., 1996). Furthermore, PP2A has been shown to regulate gene transcription by dephosphorylating the cyclic AMP-regulatory element binding protein (CREB; Wadzinski et al., 1993) and activation of c-Jun a component of the AP-1 transcription factor (Alberts et al., 1993). PP2A is highly expressed in the endothelium of developing vascular channels of the lung whereas expression in endothelium of large, muscular pulmonary arteries is barely detectable. The vasculature of the lung develops as a combination of angiogenesis and vasculogenesis (Coffin et al., 1988; Noden, 1989; Risau, 1995). Blood vessels begin to form de novo in the mesenchyme of the developing lung (vasculogenesis) by the poorly understood process whereby angioblasts proliferate and become endothelial lined vascular channels. Lung vasculature development is completed by ingrowth of large pulmonary arteries (angiogenesis) as a remnant of the sixth aortic arch with connection to the peripheral vascular tree (Noden, 1989). Although the role of PP2A in endothelial cell development is unknown, the high expression of PP2A in the endothelium of early vascular channels suggests a role for PP2A in the regulation of pulmonary vasculogenesis and not angiogenesis. This is especially intriguing as angiogenesis in the lung involves a epithelial-mesenchymal interaction between vascular endothelial growth factor (VEGF) secreting endoderm and mesodermal cells containing its receptor (VEGF-2R) similar to the model of lung epithelial development (Risau, 1995). Future studies are required to determine this potential role of PP2A in pulmonary vasculogenesis. The role of serine/threonine protein phosphatases in regulating organ morphogenesis has been clearly demonstrated in the rat kidney where okadaic acid an inhibitor of PP2A and PP1, was shown in vitro to inhibit the normal tubular branching morphogenesis of the kidney in a dose dependent manner (Svennilson et al., 1995). Importantly, the kidney shares a similar developmental origin and centrifugal morphologic pattern of development as the lung. The probes and antibodies used in the present study cannot differentiate between the a and b isoform of the catalytic subunit of PP2A therefore the results of this study reflect total catalytic subunit expression (a and b). The sequences of these two isoforms are 81% homologous in the coding region and are coded for by two separate genes. Although the a and b isoform has a similar expression pattern in adult tissues (KhewGoodall, 1988), the a isoform was always expressed at a level five to 12 times higher than the b. The physiologic significance of these two isoforms still remains to be determined. In summary, we have demonstrated PP2A developmental regulation in the lung with specific mapping of the mRNA and protein to the developing respiratory epithelium and endothelium. We speculate that PP2A is an important mediator in the complex process of epithelial development in the lung and possibly other organs such as the gut and kidney where epithelialmesenchymal interactions are important in normal organogenesis. EXPERIMENTAL PROCEDURES Tissue Preparations Sixteen time-dated pregnant Sprague-Dawley rats, ranging in gestational age from 13 to 20 days (term is 22 days), and 23 newborn rats (Hilltop Laboratory Animals, Inc., Scottsdale, PA) were sacrificed and their lungs rapidly removed. Segments of the fetal chest and neonatal lung were cut as cross or longitudinal pieces approximately 1.5 mm thick. The specimens were then immersed into fixative solution containing 4% w/v paraformaldehyde in phosphate buffered saline (PBS). After 90 min of fixation, the specimens were dehydrated in an increasing gradient of sucrose in PBS, then quickly frozen by immersion in liquid nitrogen and embedded in a 1:1 solution containing OCT compound (Miles Inc., Elkhart, IN) and 20% sucrose in PBS. Two to four µm sections were cut (at least 12 sections or six slides for each lung) and thaw-mounted onto precleaned Superfrost Plus slides (Fisher Scientific Inc., Springfield, NJ). In Situ Hybridization Sections as above were washed in PBT (PBS, 0.3% Triton X-100), digested in Proteinase K (1 µg/µl) for 10 min at 37°C, washed three times in PBT, re-fixed in 4% paraformaldehyde for 2 min, washed again in PBT, and acetylated for 10 min in 0.25% acetic anhydride, 100 mM triethanolamine HCl, and 0.09% NaCl (Sigma Chemical Co., St. Louis, MO). Sections were then dehydrated in an ethanol series (70%, 80%, 90%, 95%, 100%, and 100%), delipidated in chloroform for 15 min, washed again in 100% and 95% ethanol, and air-dried. Digoxigenin-labeled sense and antisense RNA probes were synthesized with T3 and T7 RNA polymerase (RNA Labeling Kit, Boehringer-Mannheim, Indianapolis, IN) from a template consisting of an 840 bp EcoR I/Sst I restriction fragment of the rat PP2A a isoform of the catalytic subunit (Posas et al., 1989; bp 70–910) coding region subcloned into Bluescript plasmid vector PROTEIN PHOSPHATASE 2A EXPRESSION IN DEVELOPING LUNG (Stratagene, La Jolla, CA). This coding region probe has 81% homology with PP2A b isoform of the catalytic subunit and will detect both PP2Aa and b isoforms. Subsequently the probes were hydrolyzed for 35 min at 60°C in hydrolysis buffer (80 mM NaHCO3, 120 mM Na2CO3) to give probes between 150–250 bp in length. Sections were pre-hybridized in hybridization buffer (HB; 50% formamide, 4 3 SSC, 1 3 Denhardts solution, 500 µg/ml herring sperm DNA, 250 µg/ml tRNA, 10% dextran sulfate) for 30 min at 37°C. Probes were denatured at 65°C and added to the HB at a final concentration of 1 µg/ml. Hybridization took place overnight at 37°C in a humid chamber. Slides were washed twice in 2 3 SSC, then treated with RNase A (20 µg/µl) at 37°C for 30 min, and washed again in 2 3 SSC 1 0.3% Triton X-100 three times. High stringency washes were done twice in 0.23 SSC at 42°C for 15 min each, followed by 13 SSC. Slides were rinsed in PBS, incubated for 1 hr in PBS 1 10% heat inactivated sheep serum, and then incubated for 1 hr in anti-digoxigenin antibody diluted 1:2,000 in the PBS/serum solution. Finally, slides were washed five times in PBS and two times in Genius buffer 3 (100 mM Tris-HCl, 100 mM NaCl, 50 mM MgCl2; pH 9.5), and incubated in the color substrate solution (NBT/X-phosphate plus 0.3% Triton X-100) in the dark for 24 hr. Color solution was removed by two washes in PBS, and the slides were mounted in aqueous medium (Geltol, Lipshaw, Detroit, MI) and photographed using an Olympus Vanox AHBS3 bright field microscope (Olympus, Lake Success, NY). Immunohistochemistry The immunohistochemical method employed has been previously described (Xue et al., 1996), with slight modification. After preincubating with 1% goat serum (Vector Laboratories, Burlingame, CA) for 1 hr, tissue cryostat sections were washed (2 3 10 min in PBS) and incubated at room temperature with a peptide rabbit polyclonal antibody recognizing the C-terminal portion of PP2Aa and b (Martin et al., 1994; 1:100 dilution). The control slides were incubated with PBS alone or nonspecific rabbit IgG. After removal of unbound primary antibodies by washing with PBS, the sections were incubated for 1 hr with a biotinylated anti-rabbit antibody (1:250 dilution; Vector Laboratories), followed by incubation in avidin-biotin-horseradish peroxidase complex (1:50 dilution for 45 min; Vector Laboratories). The peroxidase activity was visualized by a color reaction using diaminobenzidine (DAB; 0.5 mg/ml; Sigma) as the substrate (brown). Finally, the slides were mounted and then examined under an Olympus Vanox AHBS3 bright field microscope. Western Blot Western blotting was performed by a method described previously with minor modifications (Xue et al., 1996). Fetal lungs at day 14 (19 fetuses), 17 (27 fetuses), 19 (18 fetuses), and 20 (17 fetuses) of gestation, newborn (nine rats) and adult (90 days of age, six rats) 9 lungs were dissected, pooled and stored in 280°C. Lungs were homogenized in ice-cold 50 mM Tris-HCl buffer (pH:7.4) containing 0.1 mM EDTA, 0.1 mM EGTA, 0.1% 2-mercaptoethanol, 1 mM PMSF, 2 µM leupeptin, and 1 µM pepstatin A (Sigma). The homogenate was centrifuged at 1,000g for 10 min at 4°C and the pellet was discarded. Equal quantities of lung homogenate protein (50 µg each) and purified PP2A protein (Martin et al., 1994; 0.1 µg, provided by Dr. David Brautigan, University of Virginia, Charlottesville, VA) were loaded and separated on a 10% SDSPAGE gel, followed by blotting the proteins onto nitrocellulose (Bio-rad, Hercules, CA). Blots were subsequently stained with Ponceau Red to confirm equal loading and transfer. The blots were blocked with buffer: 50 mM Tris-HCI (pH 7.4), 0.15 M NaCl, 2% BSA, and 0.1% Tween-20, for 1 hr at room temperature. Then the blots were incubated with the anti-PP2A Cterminal polyclonal antibody (Martin et al., 1994; 1: 1,000 dilution) for 1 hr at room temperature. The blots were washed six times with PBS (5 min each) and then incubated for 1 hr with anti-rabbit IgG antibodies conjugated with horse radish peroxidase (Bio-Rad) at room temperature. The blots were washed six times with PBS (5 min each), followed by detection of immunoreactive proteins by enhanced chemiluminescence (ECL System, Amersham). Relative protein differences were determined using a densitometer (Personal Densitometer, Molecular Dynamics, Sunnyvale, CA) and analysis software (ImageQuant, Molecular Dynamics). ACKNOWLEDGMENTS The authors are grateful to Dr. David Brautigan for the gift of the PP2A antibody and Dr. Keith Norman for his critical review of the manuscript. The authors also wish to thank Mrs. Nan Zhou and Miss Tamara Stoops for technical assistance. 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