Fig. 14

Effect of treatment of diaphragms and adipocytes with sulfonylureas, PLC, PIG-P, and insulin on tyrosine phosphorylation of caveolin.

Isolated rat adipocytes (A) and hemidiaphragms (B) were incubated with increasing concentrations of Pi-specific PLC from Bacillus cereus or PC-specific PLC from Chlostridium perfringens for 40 min at 30^°C or glimepiride and tolbutamide for 2 hr at 37^°C or PIG-P or insulin for 20 min at 37^°C as indicated. Detergent-insoluble complexes were prepared from total fat-free extract of the adipocytes (A) or total membranes of the diaphragms (B), solubilized in TX-100/octylglucoside (as described in Materials and Methods), and subjected to immunoprecipitation with anti-caveolin antibodies under native conditions. The immunoprecipitates were separated by SDS-PAGE and immunoblotted with anti-phosphoty-rosine antibodies. Caveolin phosphorylated at tyrosine was detected using [¹²⁵I]protein A. The quantitative evaluations of the blot (B, each point represents the mean ± SD of four different diaphragm preparations/incubations, with caveolin im-munoprecipitations and immunoblots in duplicate) or the autoradiogram (A, representative of three separate experiments for each compound) are shown. The tyrosine-phosphorylated 21- and 24-kD proteins were identified as caveolin by probing of the same blots with anti-caveolin instead of anti-phosphotyrosine antibodies (data not shown). The amounts of caveolin recovered did not differ significantly among the different incubations. The molecular weight of the caveolin-associated 29-kD protein, which did not cross-react with anti-caveolin antibodies in the immunoblot, was derived from marker proteins run in parallel on the same gel.