Fig. 5. Co-expression of Pyk2, Cbl and ArgBP2 induces membrane ruffles and lamellipodia formation in PC12 cells, a process dependent on intact lipid rafts. (A) Pyk2, Cbl and ArgBP2 were transiently transfected into PC12-PDGFR cells. Cells were stimulated with 100 ng/ml PDGF-BB for 48 hours and subjected to immunofluorescence with goat polyclonal anti-Pyk2 (N-19), mouse monoclonal anti-Cbl (TL) and rabbit polyclonal anti-ArgBP2 antibodies. Pyk2 was stained with Alexa 488-labeled donkey anti-goat, Cbl with AMCA-labeled goat anti-mouse and ArgBP2 with TRITC-labeled swine anti-rabbit antibodies. Shown is a cell with developed lamellipodia with the focus on two different layers of the cell, as indicated by the cell drawings. The merged pictures from the three channels are also shown. (B) PC12-Myc-ArgBP2 cells, grown in the presence of 50 ng/ml NGF for 78 hours after transfection with Pyk2 and Cbl, were mock-treated (mock) or treated with 4 µM lovastatin (lovast) for 24 hours and 0.2 M methyl-ß-cyclodextrin (MBCD) for 4 hours at 37°C. Transfected cells were detected by immunofluorescence with antibodies against Pyk2 (N-19) and Cbl (C-15), which were visualized with donkey anti-goat-Alexa 488 and TRITC-labeled swine anti-rabbit antibodies. Cells showing neurites in the absence (mock) or presence of lipid raft inhibitors (MBCD/lovast) were quantified and the percentages of cells with neurites were calculated as described in Materials and Methods. Moreover, the percentage of cells showing lamellipodia at growth cones out of those cells with neurites was also determined for both untreated (mock) and treated (MBCD/lovast) cells. Error bars represent the standard error of the mean.