β3 interaction with kindlin-2, but not with c-Src, promotes angiogenic sprouting by ECs. (A) Western blots of kindlin-2 and c-Src in β3^+/+ immortalized lung endothelial cells transduced with lentivirus encoding kindlin-2 shRNA (shRNA1 and shRNA2), a c-Src shRNA, or a control (Ctrl) shRNA. β-actin was monitored as a loading control. (B) Quantification of sprouts by the cells transduced with lentiviruses encoding the kindlin-2 (upper panel), c-Src (lower panel), or Ctrl shRNAs. (C-E) Cells were transduced with kindlin-2 shRNA2 and then transiently transfected with shRNA-resistant complementary DNAs encoding either FLAG-human kindlin-2 (FLAG-hkindlin-2 WT), FLAG-hkindlin-2 QW/AA mutant, or nothing (pcDNA3.1 empty vector). (C) Western blots of human kindlin-2 detected with an anti-FLAG antibody. (D) Flow cytometry histograms depicting intracellular expression of shRNA-resistant kindlin-2 using anti-FLAG antibody. (E) Kindlin-2 rescue of endothelial cell sprout formation in the 3-dimensional fibrin gel bead assay. Note that transfection by electroporation decreased cell vitality so that the number of the sprouts was lower compared with parental immortalized lung endothelial cells. Quantification of EC sprouting is depicted as means ± SEM of 5 experiments. *P < .05; NS, not significant (paired Student t test).