Figure 6: Grp1 is essential for β1 integrin recycling.

(a) Inhibition of HGF-induced Arf6 activation by SecinH3 in iECs. After HGF stimulation of cells pretreated with SecinH3, the GTP-bound Arf6 was detected. (b) Inhibition of HGF-induced β1 integrin recycling by SecinH3 in iECs. β1 Integrin on the plasma membrane was detected as in Fig. 5a. (c) Efficacy of shRNAs for ARNO and Grp1 in HUVECs as assessed by RT–PCR. (d) Effects of cytohesin knockdown on HGF-dependent β1 integrin translocation to the plasma membrane in HUVECs. Cell surface β1 integrin was immunostained after stimulation with or without HGF. The fluorescence intensities at the plasma membrane were measured for >18 cells in each experiment. Scale bar, 40 μm. (e) Rescue of HGF-dependent β1 integrin translocation to the plasma membrane by ectopic expression of shRNA-resistant Grp1 (Grp1-res). GFP (mock) or GFP-Grp1-res was expressed in Grp1-knocked-down HUVECs (shRNA # 2 for Grp1), and cell surface β1 integrin was immunostained after stimulation with or without HGF. The fluorescence intensity of surface β1 integrin was measured for >25 cells in the lower panel. Scale bar, 40 μm. (f) Inhibition of HGF-dependent β1 integrin recycling by Grp1 knockdown in HUVECs. Internal β1 integrin after stimulation with or without HGF was immunostained with anti-β1 integrin antibody (TS2/16). The internal β1 integrin intensity was measured for >20 cells in the lower panel. Scale bar, 20 μm. (g) Inhibition of HGF-induced spreading (left panel) and FA formation (right panel) by Grp1 knockdown. HUVECs cultured on collagen type I were measured for cell area and number of paxillin puncta. (h) Co-localization of endogenous Grp1 and β1 integrin at the intracellular compartments. Grp1 (green) and β1 integrin (red) in HUVECs cultured with HGF on collagen type I were detected. Scale bar, 20 μm. Quantitative data are shown as means±s.e.m from more than three independent experiments. *P<0.05; NS, not significant, one-way analysis of variance with Dunnett’s multiple comparison test for (a,b,e) and Student’s t-test for (d,f,g).