HUVEC Lysis and Western Blotting

HUVEC cultures were trypsinized and lysed using Ripa buffer (20 mM Tris-HCl [pH 7.5], 150 mM NaCl, 1 mM Na₂ EDTA, 1 mM EGTA, 1% NP-40, 1% sodium deoxycholate, 2.5 mM sodium pyrophosphate, 1 mM β-glycerophosphate, 1 mM Na3VO4, 1 μg/mL leupeptin) containing 1× ProBlock^™ Protease Inhibitor Cocktail-50 (GoldBio) and processed as previously described[17 (link)]. Protein was then transferred to Immun-Blot PVDF Membrane at 4°C, 100 V for 1 hour 10 minutes. Blots were blocked in 2% milk proteins for 1 hour, then put in primary antibody at specified concentrations overnight. After 3 10-minute washes with PBS, secondary antibodies at specified concentrations were applied for 4 hours. After 3 additional PBS washes, blots were developed with ECL reagent. Arf6 activation assay blots were performed using commercially available kits listed in the Supplemental Information.