Pak depletion affects FA distribution. (A) Immunoblot of Pak1 and Pak2 in U2OS cells transfected for 72 h with control (noted as Ct) siRNA or with siRNAs targeting Pak1 and Pak2 (noted as Pak). The actin immunoblot was used as a loading control. (B) Immunofluorescence of paxillin and F-actin staining in U2OS cells transfected for 72 h with control siRNA (noted as Ct siRNA) or siRNAs targeting Pak1 and Pak2 (noted Pak siRNA). White boxes in the paxillin column indicate the positions of insets for higher magnifications shown in the right column. (C–E) Quantification of paxillin fluorescence intensity in the protrusion (C), the average adhesion area in the protrusion (D), and the percentage of ventral cell area containing paxillin (E) for each siRNA condition ± SEM. *, P < 1.10−6 compared with control siRNA; Student’s t test. a.u., arbitrary units. (F) Frequency histograms of paxillin foci length for control cells or cells depleted in Pak (±SEM). The numbers at the top right are the average lengths of the adhesion sites ± SEM. For quantifications in C–F, the experiment was repeated at least three times with n ≥ 30 cells for each condition, and all detectable FAs were quantified in each cell (corresponding to a minimum of 30 FAs per cell).
