FIG. 2.

Rab4 is activated via PI3-kinase- and PKC-λ-dependent pathways. After pretreatment with 50 μM LY294002, 30 μM PD98059, 50 μM myristoylated-PKC-λ pseudosubstrate, or dimethyl sulfoxide (DMSO) vehicle for 30 min (A) or 60 h after PKC-λ-expressing adenovirus infection (MOI, 30 PFU/cell) (B), cells were incubated with 17 nM insulin for 10 min. Cells were then lysed and photolabeled with [γ-³²P]GTP-azidoanilide. The samples were immunoprecipitated with the anti-Rab4 antibody, and the immunoprecipitates were resolved by SDS-PAGE. Representative results are shown at the top. Data were quantitated by a PhosphorImager as represented in the bar graph. Data represent the means ± standard errors of three independent experiments. NC, negative control (no Rab4 antibody); WT, wild type; CA, constitutively active; DN, dominant negative; Cont., control.