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Expression of two isoforms of the vacuolar-type ATPase subunit B in the zebrafish Danio rerio

S. T. Boesch, B. Eller and B. Pelster^*

Institut für Zoologie und Limnologie, Universität Innsbruck, Austria

View larger version (79K): [in a new window]   Fig. 1. Amino acid alignment (single letter code) of D. rerio (zf) V-ATPase subunits vatB1 and vatB2 (GenBank accession numbers AF472614 and AF472615), A. anguilla (eel) V-ATPase subunits vatB1 and vatB2 (Niederstätter and Pelster, 2000) and O. mykiss (trout) V-ATPase subunit isoform vatB1 (Perry et al., 2000). Yellow background color indicates identical residues, blue background color indicates most frequent residues. The origin of the sequence is indicated on the left. Possible N-glycosylation sites within the conserved region are underlined. The binding sites of antibodies 1035 and 1034 are indicated.

View larger version (60K): [in a new window]   Fig. 2. Determination of recombinant zebrafish (rZf) vatB1 and vatB2 by western blot analysis. Non-purified bacterially expressed zebrafish vacuolar ATPase subunit B isoforms vatB1 and vatB2 were probed with (A) antibody #1034, which recognizes both isoforms of the B subunit, and (B) antibody #1035, which is specific for vatB1.

View larger version (11K): [in a new window]   Fig. 3. Tissue distribution of vatB1 and vatB2. RT-PCR analysis of V-ATPase subunit B isoforms 1 and 2 in different tissues of D. rerio. 500 ng of total RNA were transcribed to cDNA using MLVV-reverse transcriptase and Random N6 Primers PCR was performed with isoform-specific primers. PCR-products were run on a 1% agarose gel and stained with ethidium bromide. Both isoforms are expressed in all tested tissues (A, vatB2 PCR product of 454 bp; B, vatB2 PCR products of 409 bp). SB, swim bladder; C, control (no template).

View larger version (55K): [in a new window]   Fig. 4. Western blot analysis of zebrafish tissues with antibodies specific for vacuolar ATPase subunit B isoforms. 30 µg of total protein were applied to each lane. Antibody #1034 recognizes both isoforms (B) whereas antibody #1035 (A) is specific for subunit isoform vatB1. Both antibodies revealed bands of approximately 55 kDa. SB, swimbladder tissue.

View larger version (92K): [in a new window]   Fig. 5. Immunocytochemical staining of vacuolar ATPase B subunit isoforms vatB1 and vatB2 in zebrafish liver. (A) No background staining was observed in the negative control without primary antibody. (B) Antibody #1035, which is specific for the vatB1 isoform, resulted in a weak staining of hepatocytes. (C) Antibody #1034, which binds to vatB1 and vatB2, resulted in a very strong signal in most hepatocytes. Scale bars, 50 µm.

View larger version (96K): [in a new window]   Fig. 6. Immunocytochemical staining of vacuolar ATPase B subunit isoforms vatB1 and vatB2 in the small intestine of the zebrafish. (A) No background staining was observed in the negative control without primary antibody, except in the region of the microvilli, which contains levamisol-resistant alkaline phosphatase. (B) Antibody #1035, which is specific for the vatB1 isoform, resulted in a weak staining of cells. The microvilli showed unspecific staining, and some cells in the central region of the villi near the blood vessels of the lamina propria showed a positive reaction. (C) Antibody #1034, which binds to vatB1 and vatB2, resulted in a very strong signal in large cells at the base of the villi, which according to their position and shape most likely represent neurosecretory cells. mc, mucus cells; lp, lamina propria. Arrows identify large positive cells. Scale bars, 50 µm.

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