Figure 7

IQGAP1 and caveolin-1 are upstream and downstream scaffolds in the same ERK1/2 activation pathway. (A) Double knock down of IQGAP1 and caveolin-1 shows the same decrease in DPBA-induced ERK1/2 activation as single knock down. Cells were transfected with siRNA as indicated, then stimulated with DPBA for 5 minutes. Lysates were subjected to western blotting for analysis of ERK1/2 phosphorylation. (B) C-Raf activation is reduced after knock down of caveolin-1, but not after knock down of IQGAP1. Cells were transfected as indicated, then stimulated with DPBA for 5 minutes. The ratio of phospho-C-Raf (S338) to total C-Raf was analyzed on duplicate membranes after normalization to GAPDH. (C-E) To enrich the cytoskeletal fraction, DPBA stimulated A7r5 cells were subjected to Triton X-extraction before preparation of soluble and insoluble cell extracts. Insoluble and soluble lysates were analyzed for ERK1/2 phosphorylation as well as target protein expression by western blotting and densitometry. Data represent five independent experiments. (C) Western blots show siRNA knock down of caveolin-1, IQGAP1, B-Raf and C-Raf. Insoluble samples are shown for caveolin-1 and IQGAP1 expression, soluble samples are shown for B-Raf, C-Raf and GAPDH. (D) The graph shows average ERK1/2 phosphorylation in the TX-insoluble fraction, along with a representative western blot. (E) The western blot shows phosphorylated ERK1/2 and total ERK1/2 in TX-soluble samples. Significance compared to control (*) and compared to IQGAP1 siRNA (#), as well as p values are indicated on graphs; error bars represent standard errors.