Pak activity is regulated by adhesion strength. (A) Cell lysates from PtK1 cells plated on low (5 µg/ml, FN5), medium (10 µg/ml, FN10), or high (30 µg/ml, FN30) FN concentrations were analyzed by immunoblotting and probed with phospho-specific antibodies anti-Pak1/Pak2 (active Pak1/Pak2) and antibodies to total proteins anti-Pak1/Pak2 and anti-actin. Two exposure times of the same immunoblot are shown: long (2-min exposition) and short (15-s exposition). Quantification of immunoblots was determined by the ratio of phosphorylated to total Pak1/Pak2 and was normalized to the FN10 condition, which correlates with optimal cell migration. The values shown are averages of three independent experiments. (B) Immunofluorescence with antibody directed against active Paks (pPak), paxillin, and F-actin phalloidin staining in PtK1 cells plated on FN5, FN10, and FN30. White boxes in the merge column indicate the positions of insets for higher magnifications shown in the rightmost column. (C) Frequency histogram of pPak fluorescence intensity in FAs for each FN concentration (low FN5, medium FN10, and high FN30). The numbers at the top right are the average pPak intensities ± SD. The data shown are representative of one experiment and are averaged from n ≥ 30 cells for each condition. The experiment was repeated three times with similar results. All detectable FAs were quantified in each cell (corresponding to a minimum of 30 FAs per cell). The red arrows indicate the increased frequency of FAs containing a low pPak level observed in cells plated at a high adhesion strength (FN30). In contrast, the black arrow indicates the increased frequency of FAs containing a high pPak level observed in cells plated at a low adhesion strength (FN5).