Reduced accumulation of phosphorylated FAK and Pxn in focal adhesions of cells with impaired RSF assembly. (A) Immunolocalization of F-actin, Pxn, and phosphorylated FAK (FAK-pY397) in representative WT, Dia Inh, and Atn-1 KD U2OS cells. The bottom row shows images representing the ratio of background-subtracted ratio intensity of FAK-pY397 to Pxn at individual focal adhesions in each of the three conditions. The range of the ratio is indicated by the bottom-right scale (in arbitrary units). Bar, 5 µm. (B) Bar graph of the mean ratio of FAK-pY397:Pxn, normalized to the WT cells (error bars indicate SEM; n > 1,000 focal adhesions from 10 cells for each condition; ***, P < 0.001 with respect to WT). (C) The mean intensity (Inten.) of FAK-pY397 or Pxn plotted against the mean intensity of actin at each focal adhesion (FA) for the focal adhesions shown in the bottom row of A. A.U., arbitrary unit. (D) Immunolocalization of actin, Pxn, and phosphorylated Pxn (Pxn-pY31) for each condition. The bottom row of images indicates the ratio of Pxn-pY31 to Pxn for each condition described. The range of the ratio is indicated by the bottom-right scale (in arbitrary units). Bar, 5 µm. (E) Bar graph of the mean ratio of Pxn-pY31:Pxn, normalized to the WT cells (error bars indicate SEM; n > 700 focal adhesions from six cells for each condition; **, P < 0.01 with respect to WT). (F) The mean intensity of Pxn-pY31 or Pxn plotted against the mean intensity of actin at each focal adhesion for the focal adhesions shown in the bottom row of D.