Extended Data Fig. 6: CD82 sustains RhoA activity in ECs.
(a) HUVECs transfected with CD82 or control siRNA were serum-starved overnight, stimulated with VEGF-A (50 ng/ml), and then lysed with RIPA buffer. GTP-bound RhoA was pulled down from the lysates with GST-Rhotekin RhoA-binding domain fusion and detected in Western blot. Other indicated proteins in the lysates were directly examined with Western blot. The ratios of GTP-bound RhoA/total RhoA were quantified based on the band densities of RhoA (mean±SD; n=3 individual experiments). (b) Localization of GFP-AHPH. MLECs cultured on coverslips were transfected with GFP-AHPH WT or GFP-AHPH A470D&E758K mutant and processed for confocal microscopic imaging. (c) CD82 silencing up-regulates p190RhoGAP activity in ECs. HUVECs transfected with CD82 siRNAs or control siRNA were serum-starved overnight and then stimulated with VEGF-A (50 ng/ml). After cell lysis with a lysis buffer containing 1% Triton X-100, the p190RhoGAP proteins in the lysates were immunoprecipitated and then immuno-blotted with phosphotyrosine mAb (4G10) and p190RhoGAPmAb. Other indicated proteins in the lysates were directly examined with Western blot. (d) Immunofluorescence staining of VE-cadherin and Rnd3 in the MLEC monolayers. Images were acquired with confocal microscopy. (e) Western blot analysis of Rnd3 in the HUVECs that were transfected with control siRNA and Rnd3 siRNA. (f) Histochemistry analysis (H-E staining) on the effects of CN04 and/or Y27632 on LPS-induced acute lung injuries in WT C57BL/6 mice.
