Fig. 5.

Cav-1 Tyr14 is required for Cdc42-Cav-1 and βPix-Cdc42 complex formation. A: MIN6 cells were transiently transfected with RhoGDI-HA or Myc-tagged Cav-1 DNAs (WT or Cav-Y14F). Cells were preincubated in MKRBB for 2 h, glucose-stimulated for 2 min, and the resultant cleared detergent cell lysates were immunoblotted to validate expression of recombinant proteins, relative to endogenous Cdc42. Lysates were otherwise subjected to anti-βPix immunoprecipitation (IP) reactions (B) or GST-Cdc42-GDP interaction assays (C), as described in materials and methods. Precipitates were resolved on 12% SDS-PAGE, transferred to PVDF, and immunoblotted (IB) for βPix, Cdc42, Myc (Cav-1), and GST. Reassembly of noncontiguous lanes from within the same gel is demarcated by black lines. Data shown are representative of 3–5 independent experiments.