Analysis of FA turnover. (A) IF of p14^f/− and p14^−/− MEFs with anti-Paxillin and anti-Zyxin antibodies. White arrows indicate leading edge formation areas in the control cells where no Zyxin-positive FAs are detected (magnification). In the p14 knockout MEFs the overall elongated FAs are also positive for Zyxin (magnification). (B) FRAP analysis of FAs in indicated MEFs. Image shows representative example of FAs (white arrows) before bleaching and during recovery time in p14^f/− MEFs. Graph shows curves of fluorescence recovery (mean of 10 cells per group, number of measured FAs = 60) after bleaching of indicated MEFs. (C) TIRF microscopy of MEFs expressing mRuby-Paxillin. See also Videos 2 and 3. Shown are mature FAs of magnified areas of control and p14 knockout MEFs at three different timeframes (1 frame = 10 min). Merging of the three color-coded frames indicates the development of single FAs over time. Note: white FAs are stable over the shown time period (20 min). (D) Quantification of FA turnover. The left graph displays the existence of single FAs (n = 168) as dots over a time period of 60 min. All existing FAs were included. The right graph shows dynamic FA events. New growing FAs were analyzed during a time period of 80 min (P < 0.001).