Figure 6.

Re-introduction of ARF exon 2 encoded domain in ARF-depleted cells restore both spreading and pFAK expression. Rescue experiment was performed as described before, except that siRNA for the 5’-UTR of ARF gene was used (siRNA-3), and different ARF tagged forms were tested. (a) Scheme of N-ter flagged forms of ARF used in this study. (b) Immunoblot analysis of flagged 14 ARF mutants and tubulin (loading control) expression levels in whole-cell lysates of cells transfected and silenced with the indicated plasmids and siRNAs. Asterisks indicate an aspecific flag band. (c) Quantification of spreading efficiency of cells. Data were quantified from three or more experiments and 400 cells were analyzed for each transfection point in every experiment. Error bars represent the s.d. from the mean and statistical analysis performed as described in Figure 5. *P<0.02 siARF-empty vector vs siSCR-empty vector; **P<0.0092 siARF-Flag-ARF vs siARF-empty vector; ***P<0.0112 siARF-Flag 65–132 vs siARF-empty vector. (d) Analysis of pFAK and total FAK intracellular protein levels upon rescue experiment by IP with anti-t-FAK antibody and IB with anti-pFAK Y397 antibody performed as previously described. Representative western blots are shown. pFAK band intensities normalized versus t-FAK are shown below each corresponding band, and are expressed as fold value with respect to empty vector siSCR-transfected sample, arbitrarily set to 1 (see Materials and Methods section for details).