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Fig. 2. Antibody characterization and ClC-3 expression in mouse B-cells. Immunoblots of mouse islet homogenate and cells overexpressing ClC-3 isoforms. (A) Whole-cell lysates (10 µg/lane), crude membrane and cytosol isolates (100 µg/lane) were prepared from tsA cells stably transfected with full-length hClC-3 or vector alone (mock), or from mouse islets. Anti-hClC-3c detected a 120 kDa protein in the membrane fractions of the transfected cells. After deglycosylation with N-glycanase for 18 hours immunoreactivity was detected as a single band at {approx}90 kDa in the tsA cells. In islets, both the glycosylated form (double band at 115-130 kDa) as well as the deglycosylated protein ({approx}90 kDa) were detected. (B) Isoform specificity of anti-hClC-3c was verified by immunoblotting of deglycosylated whole-cell lysates (10 µg/lane) obtained from HEK 293 cells stably expressing hClC-3, hClC-1, hClC-4 or rat ClC-2. (C) and (D), as in (A) and (B), except that anti-hClC-3n was used.