β3 regulates phosphorylation of VASP. (A) WT, β3−/−, or β3-GFP cells either untreated or treated with 1 µM H89 (PKA signaling inhibitor) or 1 µM forskolin (PKA signaling activator) were analyzed by Western blotting for total VASP, phospho-S153 VASP, or total Mena. WT and β1−/− cells were lysed and analyzed by Western blotting for total VASP. (B) Quantification of blots from WT, β3−/−, or β3-GFP cells shown in A. Data are mean ± SEM from four independent experiments (indicated by error bars). ***, P < 0.001 compared with WT control levels. (C) Confocal images of WT and β3−/− cells plated on 2D FN (left; the dotted lines indicate the edge of the cells) or CDM (right) and stained for VASP (FITC, green), vinculin (Alexa Fluor 568, red), and FN (CDM only, Alexa Fluor 633, blue). Merged, enlarged images are shown. Bars: (left) 5 µm; (right) 10 µm. (D) WT and β3−/− cells transfected with WT-VASP-GFP or S153A-VASP-GFP and subjected to FRAP analysis. τ1/2 values (s) were obtained from normalized recovery data for each construct in each cell type. Data are shown as mean values ± SEM (indicated by error bars) from at least 10 cells from four independent experiments. **, P < 0.01; *, P < 0.05 compared with WT VASP recovery in WT cells. (E) WT and β3−/− cells transfected with talin-GFP or vinculin-GFP and subjected to FRAP analysis. τ1/2 values (s) were obtained from normalized recovery data for each construct in each cell type. Data are shown as mean values ± SEM from at least 11 cells from three independent experiments. **, P < 0.01 compared with recovery in WT cells. (F) FRAP analysis of talin-mCherry recovery at GFP-positive adhesions in β1-GFP or β3-GFP cells. τ1/2 values (s) were obtained from normalized recovery data for each construct in each cell type. Data are shown as mean values ± SEM from at least 14 cells from four independent experiments (indicated by error bars). **, P < 0.01 compared with recovery in β1-GFP cells.
