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. 2010 Mar 22;188(6):877–890. doi: 10.1083/jcb.200906012

Figure 6.

Figure 6.

Myosin II promotes tyrosine phosphorylation and interaction of paxillin, vinculin, and FAK. (A) Immunoblot of lysates of untreated MEF cells (control) and cells treated with 20 µM blebbistatin (Blebb) using antibodies specific to paxillin (Pxn) or pY31, pY118, or pS273 paxillin (PxnY31, PxnY118, and PxnS273). Numbers above each blot represent the mean of quantified Western blots ± 95% confidence interval (n = 4 experiments). (B) Immunolocalization of paxillin (green) and pY31 or pY118 paxillin (red) in untreated control cells and cells treated with 20 µM blebbistatin. The third columns show merged images Bar, 2 µm. (C) Effects of blebbistatin on adhesion localization of pY31 and pY118 paxillin in adhesions, expressed (also in F) as the ratio of mean fluorescence intensities within segmented adhesions of 20 µM blebbistatin-treated relative to control cells. Antibody competition for similar epitopes precluded comparison with effects of blebbistatin on total paxillin level in the same cells. Mean fluorescence ratio is shown above each plot. n = number of blebbistatin-treated cells/number of control cells (also in F). (D) IP with antibodies to paxillin (left and top right) or Crk (bottom right) from lysates of untreated control MEFs or MEFs treated with 20 µM blebbistatin followed by immunoblotting with antibodies to paxillin, PY epitopes (P-Tyr), pY31 paxillin, FAK, β-Pix, or Crk. Numbers above each blot represent the mean of quantified Western blots ± 95% confidence interval (n = 3 experiments). (E) Immunofluorescence analysis of untreated control cells or cells treated with 20 µM blebbistatin using antibodies specific to pY397 FAK (red) or PY epitopes (green). Merged images are shown in the third column. Bar, 2 µm. (F) Effects of 20 µM blebbistatin on adhesion localization of pY397 FAK and PY in adhesions quantified from immunofluorescence images. *, P < 0.02. (G) Immunoblot of lysates of untreated MEF cells, cells treated with 20 µM blebbistatin, or a combination of 20 µM blebbistatin and either 10 µm PP2, 10 µm PF271 (FAKi), or all three using antibodies specific to paxillin, pY31, pY118 paxillin, FAK, pY397 FAK, Src (c-Src), or pY527 Src (cSrcY527). Error bars indicate 95% confidence interval of the mean.