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Localization of the vacuolar-type ATPase in swimbladder gas gland cells of the European eel (Anguilla anguilla)

S. T. Boesch, H. Niederstätter^* and B. Pelster

Institut für Zoologie und Limnologie, Universität Innsbruck, Austria
^* Present address: Institut für Gerichtliche Medizin, Universität Innsbruck, Müllerstr. 44, A-6020 Innsbruck, Austria

View larger version (69K): [in a new window]   Fig. 1. Expression of vacuolar ATPase subunit B isoforms vatB1 and vatB2 in BL21(DE3)pLysS cells: bacterial lysates have been separated by SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) and stained with Coomassie blue. Expression was induced with 1 µmol l-1 IPTG (isopropylthiogalactoside). The expression of recombinant protein was clearly induced after 2 h, and the expression increased with time (e.g. after 4 h). Arrows indicate the position of reference vatB1 and vatB2 bands at approximately 60 kDa.

View larger version (16K): [in a new window]   Fig. 2. Determination of specificity of the antibodies by western blot analysis with non-purified bacterially expressed eel vacuolar ATPase subunit B isoforms vatB1 and vatB2. 30 µg protein were used for each lane. (A) Western blot probed with antibody #1035, which is specific for vatB1: left lane, vacuolar ATPase B subunit isoform vatB1 (kidney isoform); right lane, vacuolar ATPase B subunit isoform vatB2 (brain isoform). (B) Western blot probed with antibody #1034, which recognizes both isoforms of the B subunit. Without primary antibody, no bands could be detected.

View larger version (41K): [in a new window]   Fig. 3. Western blot analysis of eel swimbladder tissue with antibodies specific for vacuolar ATPase subunit B isoforms. 30 µg of swimbladder protein were used for each lane. Antibody #1034 recognizes both isoforms (rabbit anti-vatB1+vatB2), whereas antibody #1035 (rabbit anti-vatB1) is specific for subunit isoform vatB1. Both antibodies revealed bands of approximately 55 kDa. Without primary antibody, no bands could be detected.

View larger version (67K): [in a new window]   Fig. 4. Immunocytochemical staining of vacuolar ATPase B subunit isoforms vatB1 and vatB2 in swimbladder gas gland cells from Anguilla anguilla. Both antibodies revealed positive staining only in gas gland epithelial cells. (A) Antibody #1034 (rabbit anti-vatB1+vatB2) revealed a positive staining of apical and basolateral membranes. A very intense signal was also observed in apical vesicles. (B) Staining with antibody #1035, which is specific for the vatB1 isoform (kidney isoform), resulted in a similar picture in the apical region of the cells, but in the basolateral region very little staining was observed. No background staining was observed in the negative control (C). Scale bars, 10 µm. Arrows point to positive staining reaction in basolateral membranes.

View larger version (109K): [in a new window]   Fig. 5. Immunocytochemical localization of surfactant protein D (SP-D) in swimbladder gas gland cells of Anguilla anguilla. The antibody directed against human SF-D revealed staining especially of vesicles located in the apical region of the cells. Arrowheads indicate stained lamellar bodies in the gas gland cells. Scale bar, 10 µm.

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