Paxillin Y31/118 phosphorylation is sufficient for promoting paxillin–vinculin interaction and labile vinculin recruitment to adhesions. (A) Immunoblot analysis of lysates of untreated (control) and cells treated with 20 µM blebbistatin (Blebb) or with 20 µM blebbistatin and 100 µM Na₃VO₄ using antibodies specific to paxillin (Pxn), pY31 paxillin, pY397FAK, or FAK. (B) Comparison of cells treated with 20 µM blebbistatin or 20 µM blebbistatin and 100 µM Na₃VO₄ and immunolabeled with antibodies to paxillin (red) and vinculin (Vcl; green). Bar, 2 µm. (C) Effects of Na₃VO₄ on vinculin localization in adhesions of blebbistatin-treated cells, shown (also in G and H) as the ratio of mean fluorescence intensities within segmented adhesions of 20 µM blebbistatin-treated cells relative to non–blebbistatin-treated cells in the presence and absence of additional Na₃VO₄. The mean fluorescence ratio is shown above each plot. (D) Antipaxillin IPs from lysates of untreated control or cells treated with either 20 µM blebbistatin, 100 µM Na₃VO₄, and 20 µM blebbistatin or 100 µM Na₃VO₄ alone followed by analysis by PAGE and immunoblotting with antibodies to vinculin, paxillin, or PY epitopes. (E–I) EGFP-conjugated paxillin (Pxn-GFP wt) or paxillin bearing mutations of tyrosines 31 and 118 to phenylalanines (Pxn Y31/118F) or glutamic acids (Pxn Y31/118E) were expressed in MEFs and either treated with 20 µM blebbistatin or not. (E) Images of cells expressing EGFP-conjugated paxillin mutants (green) and immunolocalization of vinculin (red) or PY epitopes (P-Tyr; red). Right columns show (also in F) merged, magnified images of the boxed regions Bar, 2 µm. (F) Images of EGFP-conjugated Y31/118E paxillin (green) and immunolocalization of zyxin (Zyx; red) or α-actinin (Actn; red) in cells treated with 20 µM blebbistatin. (G) Effects of blebbistatin on adhesion localization of vinculin and paxillin in cells overexpressing wild type (WT) or mutant (Y31/118E) paxillin-EGFP. (H) Effects of blebbistatin on adhesion localization of zyxin, α-actinin, and paxillin in cells overexpressing Y31/118E paxillin-EGFP. (I) FRAP analysis of mCherry-vinculin. mCherry-vinculin was expressed alone (control) or together with EGFP conjugates of wild-type or Y31/118E paxillin in untreated cells or cells treated with 20 µM blebbistatin (+Blebb), and FRAP was performed of the mCherry vinculin fraction in adhesions. Half-times of mCherry vinculin fluorescence recovery. Means are shown above each plot. n = number of adhesions. (J) Immunoblots of lysates of blebbistatin-treated (B) or untreated (C) cells that had been mock transfected or transfected with the EGFP-paxillin mutants and probed with antibodies to paxillin, pY31 paxillin, or tubulin. (K) Anti-GFP immunoprecipitates from MEFs expressing EGFP-tagged paxillins and either untreated (C) or treated with 20 µM blebbistatin (B) and probed by immunoblotting with antibodies to vinculin and GFP. Quantification of blots is shown below. WB, Western blot. *, P < 0.02. Error bars indicate 95% confidence interval of the mean. (C, G, and H) n = number of blebbistatin-treated cells/number of control cells.