Supplementary Figure 8

(a) Immunofluorescence of FL-Kank2-GFP (green) co-stained with endogenous RhoGDIα (red) and DAPI (blue). Scale bar, 10 μm. (b) Western blot (left) and densitometric (right) analysis of RhoA activation in Kank2-depleted cells expressing indicated Kank2 constructs seeded on FN for 1h (mean ± s.d.; n = 3 independent experiments; P values were calculated using one-way ANOVA Tukey test; N.S., no significance). (c) Western blot (left) and densitometric (right) analysis of myosin light chain (MLC) phosphorylation at serine-19 (pMLC-Ser19) in Kank2-depleted cells expressing indicated Kank2 constructs seeded on FN for 1h. Actin was used to control protein loading (mean ± s.d.; n = 4 independent experiments; P values were calculated using one-way ANOVA Tukey test; N.S., no significance). (d) Still images from a representative time lapse FRAP experiment of Talin-1-GFP shown in rainbow LUT. Talin-1-GFP was co-expressed with Cherry-tagged FL-Kank2 or Kank2ΔKN, respectively, in Kank2-deplted fibroblasts. Co-localization of Kank2-mCherry and Talin-1-GFP in the region of interest (ROI, red circle) was confirmed in pre-bleached images. Scale bar, 2 μm. Source data for b,c can be found in Supplementary Table 2.