Loss of p14–MP1 endosomal signaling impairs cell migration and FA. (A) Migration of MEFs in wound-healing assay. Red lines depict edges of the wound. See also Video 1. (B) Calculation of migration speed of MEFs in wound-healing assay (µm/h, mean of cell migration speeds ± SD; **, P < 0.001, Student’s t test). (C) IF: anti-Paxillin antibody. Note formation of elongated peripheral FAs in p14^−/− MEFs (red arrows) compared with FAs and FCs at the leading edge of the control p14^f/− MEFs (red arrowheads). Expression of myc6-MP1 or Xpress-p14-CAAX does not restore FA pattern in p14^−/− MEFs (presence of myc6-MP1 is confirmed by anti-myc antibody showing cytoplasmic distribution of MP1, presence of p14-CAAX is confirmed by anti-Xpress antibody). Reduction of FA size can be achieved by expression of p14-GFP (blue arrowheads, presence and localization of p14-GFP is confirmed by GFP image). Bar, 10 µm. (D) Quantification of average FA length and detailed analyzes of FA populations (“Length distribution”) in MEFs. Left graph: mean in percent ± SEM compared with control p14^f/− MEFs (mean of FA length in control p14^f/− MEFs was taken as 100%). Right graph: FAs are clustered in four specified length groups (x-axis) and are presented in percentage of all adhesions (y-axis) in each type of MEFs. See also Table S1. (E) FACS analysis of activated β1 integrins (identified with 9EG7 antibody) reveals no difference in expression in p14^f/− MEFs (black line) and p14^−/− MEFs (blue line; dotted lines indicate negative controls). The data shown are from a single representative experiment out of three independent experiments. For complete integrin profile, see Fig. S2.