FIG. 5.

Caveolin 1 is sufficient and required to alter PI(3)K activity in response to ceramide. (A) Rat-1 fibroblasts were treated with 50 μM C2-ceramide for the indicated times, followed by immunoprecipitation as indicated. Immunoprecipitations were split, and half were used for PI(3)K assays (top three panels) and the other half were subjected to SDS-PAGE, immunoblotting as indicated, and fluorimage scan (bottom two panels). (B) Rat-1 fibroblasts were either mock transfected or transfected with HA-caveolin or an empty HA vector. After selection, cells were lysed and split for PI(3)K assay (top panel), α-HA Western blotting (middle panel), or immunoprecipitation and blotting as indicated (bottom panel). (C) Rat-1 fibroblasts were infected with an antisense caveolin 1 construct or antisense caveolin 3 followed by C2-ceramide or null treatments and lysis. Lysates were split, and half were used for PI(3)K assays (bottom two panels of each box), and the other half were subjected to SDS-PAGE, immunoblotting as indicated, and fluorimage scan (top two panels of each box).