Mini protean tgx precast 7.5 gel

Transduced ECs were starved for 3 h with EBM-2 medium (Lonza) at 37 °C, 5% CO₂ in a humidified atmosphere. Cells were surface labeled with the cell impermeable HaloTag PEG-Biotin Ligand 3 µM at 4 °C for 15 min, stimulated or not with VEGF-A165 30 ng/ml for 10 min at 37 °C. ECs were lysed in buffer containing 25 mM Tris–HCl pH 7.6, 100 mM NaCl, 1% Triton X-100, 5% glycerol, 0.5 mM EGTA, 2 mM MgCl₂, 1 mM PMSF, 1 mM Na₃VO₄ and protease inhibitor cocktail. Cellular lysates were incubated for 20 min on wet ice, and then centrifuged at 15,000 × g, 20 min, at 4 °C. The total protein amount was determined using the bicinchoninic acid (BCA) protein assay reagent (Pierce). Equivalent amounts (1 mg) of protein were immunoprecipitated for 2 h at 4 °C on streptavidin-conjugated beads. Immunoprecipitates were washed three times with lysis buffer with or without detergent and then separated by SDS–PAGE with Mini PROTEAN TGX precast 7.5% gel (BIO-RAD). Proteins were then transferred to a Trans-Blot Turbo™ PVDF Transfer (BIO-RAD), probed with antibodies of interest and detected by enhanced chemiluminescence technique (PerkinElmer).