Category Archives: Isoforms

Localization of the a Subunit of the V-ATPase: a1, a2, and a4 Isoforms Isoforms al and a4 were detected in the apical pole of narrow cells (data not shown) and in clear cells of the epididymis (Fig. 7, A and B) and vas deferens (data not shown). Double labeling for the E2 subunit (Fig. 7C) revealed that al colocalized with E2 in subapical vesicles but was absent from the microvilli (arrows in Fig. 7, A, C, and E). Subunit a4 showed a complete colocalization with E2 in subapical vesicles and microvilli of clear cells (Fig. 7, B, D, and F). Expression of the osteoclast-specific a3 isoform was not examined. (more…)

However, the cytosolic staining for Cl seemed stronger compared to that of C2. Double labeling for the E2 subunit (Fig. 5, B, B, E, and E ) revealed colocalization of both C isoforms with E2 in subapical vesicles (Fig. 5, C, C0, F, and F0; yellow staining). In contrast, Cl and C2 were barely detected in apical microvilli (Fig. 5, C, C0, F, and F0; red staining). No significant staining was detected in principal cells. (more…)

Similarly to narrow cells, clear cells were identified by their coexpression of the E2 subunit (Fig. 3B). A weak cytosolic staining was also detected in these cells, as we have previously reported for other subunits of the V1 domain of the pump, including subunit E2. Figure 3C shows a complete colocalization of subunits A and E2 in subapical vesicles and apical microvilli (yellow staining). Immunogold electron microscopy confirmed the localization of subunit A in apical microvilli in addition to its subapical localization (Fig. 3D). Interestingly, this subunit was also detected in intracellular structures of principal cells located in the proximal regions of the epididymis (Fig. 4A). (more…)

Specificity of the V-ATPase Antibodies Each of the V-ATPase subunit antibodies used in this study, except the novel anti-subunit A antibody, has been characterized previously. The specificity of these antibodies in epididymis samples was further confirmed by Western blotting. As shown in Figure 1, all antibodies gave one single band at the appropriate molecular weight, showing their purity. A more complete analysis was performed for the novel anti-A antibody by repeating the Western blot after preincubation of the antibody with the immunizing peptide. (more…)

Afterthree washes in TBS with 0.1% Tween 20 and 15-min block in TBS with milk, membranes were incubated with a donkey anti-rabbit IgG conjugated to horseradish peroxidase (Jackson ImmunoResearch Laboratories) for 1 h at room temperature. After five further washes, antibody binding was detected with the Western Lightning Chemiluminescence reagent (Perkin Elmer Life Sciences, Boston, MA) and Kodak X-Omat blue XB-1 films. (more…)

Protein Extraction and Western Blotting Epididymis was harvested from anesthetized rats. Tissue was cut into small pieces and rinsed several times in PBS/protease inhibitors to remove most of the sperm. Tissue was homogenized in 10 ml/g of buffer containing 250 mM sucrose, 18 mM Tris, 1 mM EDTA, and complete protease inhibitor (Roche Applied Science, Indianapolis, IN), adjusted to pH 7.4 with HEPES, using a PRO 200 homogenizer followed by 20 strokes in a glass potter fitted with a Teflon pestle (Thomas Scientific, Swedesboro, NJ). (more…)

Sections were washed in PBS (3X for 5 min) and then blocked in PBS containing 1% BSA for 15 min. Primary antibodies were applied in a moist chamber for 90 min at room temperature or overnight at 4°C. Sections were washed in high-salt PBS (2.7% NaCl) twice for 5 min and once in normal PBS. Secondary antibody was then applied for 1 h at room temperature followed by washes as described above. Slides were mounted in Vectashield medium (Vector Laboratories, Inc., Burlingame, CA). Some sections were double-stained by subsequent incubation with another primary antibody raised in a different species, followed by an appropriate secondary antibody. (more…)

Immunofluorescence: Conventional and Confocal Microscopy Sprague-Dawley rats (Charles River Laboratories, Wilmington, MA) were acquired, retained, and used in compliance with the National Research Council’s recommendations. Sexually mature male rats were anesthetized with Nembutal (0.5 ml i.p.; Abbott Laboratories, North Chicago, IL) and perfused via the left ventricle with PBS (0.9% NaCl in 10 mM sodium phosphate buffer, pH 7.4) followed by fixative containing 4% paraformaldehyde, 10 mM sodium periodate, 75 mM lysine, and 5% sucrose in 0.1 M sodium phosphate buffer (PLP), as described previously. (more…)

Antibodies Affinity-purified rabbit polyclonal antibodies against the V-ATPase C1,C2, G1, G3, a1, a2, a4, d1, and d2 subunit isoforms were used. These antibodies have been characterized previously. An affinity-purified polyclonal antibody raised in chicken against the V-ATPase E2 subunit was also used to identify narrow and clear cells. A novel affinity-purified rabbit polyclonal antibody against the last 10 amino acids (CMQNAFRSLE) of the C-terminal tail of the V-ATPase A subunit was also used and was characterized in this study. (more…)

Four a subunit isoforms have been identified in the human V-ATPase: al, a2, a3, and a4. Subunit al is expressed ubiquitously; subunit a2 was detected in the kidney, lung, and spleen; and subunit a3 was localized in osteoclasts, where it is essential for bone resorption. Subunit a4 was detected in the kidney, inner ear, and the murine epididymis. Two d subunit isoforms have been identified for the human V-ATPase: dl is ubiquitous, whereas d2 is present in kidney, lung, and osteoclast. (more…)