PDGF-BB–stimulated integrin β3 macropinocytosis is BARS dependent but clathrin and CAV1 independent. (A) BARS, CHC, CAV1, and WAVE1 protein expression levels from whole-cell lysates were analyzed by SDS-PAGE and Western blotting. (B and C) siRNA-transfected primary mouse fibroblasts were stimulated with PDGF-BB for 10 or 15 min, fixed, and IF stained. The number of cells forming integrin β3 CDRs or macropinosomes per 100 cells was counted (n = 5). (D) siRNA-transfected primary mouse fibroblasts were prepared as in Fig. 2 C. (E) Quantified from the flow cytometry data in D, the percentages of cell surface integrin β3 mean fluorescence intensity (MFI) decrease after 15-min PDGF-BB stimulation compared with no stimulation are plotted (n = 3). (F) siRNA-transfected primary mouse fibroblasts were prepared as in Fig. 2 D. Arrowheads denote internalized integrin β3 at macropinosomes. Arrows denote recycled integrin β3 at focal adhesions. (G) The BARS siRNA-transfected cell in Fig. S2 A was stimulated with PDGF-BB. The temporal and spatial translocation of integrin β3–GFP was traced by 4D time-lapse confocal live-cell imaging as in Fig. 1 D. Arrows in the t = 0 min image denote the integrin β3–GFP at original focal adhesions; arrows in t = 28 min and 60 min images point at the same positions to denote the disappearance of integrin β3–GFP at focal adhesions without integrin β3–GFP recycling to form new focal adhesions. Arrowheads in t = 5 min, 8 min, and 10 min images denote the integrin β3–GFP at a CDR that did not fully close up or sink into the cytosol to form macropinosomes because of the lack of the BARS protein. Error bars represent means ± SD. ***, P < 0.05. Bars: (F) 100 µm; (G) 20 µm.