Figure 2.

ARF interacts with active FAK during adhesion. ARF colocalizes with active FAK (phosphorylated FAK on Tyr397) and phosphorylated Paxillin during adhesion. HeLa cells were allowed to adhere onto fibronectin-coated coverslips for 5 and 24 h as described before, fixed and subjected to IF with anti-ARF and either pFAK Y397 antibody or pPaxillin Tyr118 antibody. Representative images showing ARF and pFAK (a) or ARF and pPaxillin (b). Subcellular localization are shown for each time point. Images were taken as described before (scale bar, 10 μm). Histograms below each panel, representing the mean of three independent experiments, reports the percentage of cells in which ARF colocalize with pFAK or pPAXillin Tyr118. S.d. are also shown. Asterisks indicate statistically significant differences (P<0.001) as described in Figure 1. ARF and FAK co-immunoprecipitation. (c) Cytoplasmic HeLa extracts collected upon cell detachment (lanes 1–2), during spreading (lanes 3–4), and 24 h after adhesion (lanes 5–6) were subjected to immunoprecipitation with anti-ARF antibody or IgG as negative control and western blot with FAK and ARF antibodies. Inputs probed with anti-FAK antibody are shown. (d) Same experiment described in c performed on HaCaT cells except that IB was performed with anti-pFAK Y397. Molecular weights of both ARF and FAK are shown on the left of each western blot.