Ab74012

Protein was extract from HUVEC and HMEC cells that were either grown in complete medium or starved for 24 h in serum-free culture medium before exposure to a 300 mV/mm direct current electric field in vitro. After electric field stimulation cells were rinsed with cold PBS and lysed with lysis buffer CelLytic-M (C2978; Sigma) containing protease inhibitors (11836170001, complete mini EDTA-free, Roche) and phosphatase inhibitors (4906837001, PhosSTOP, Roche). Ten micrograms of total protein were electrophoresed by 12% SDS-PAGE at 100 Volts for 2.5 h at room temperature, and transferred onto nitrocellulose membrane (Z613630, Sigma) using the Bio-Rad system at 100 Volts for 1.5h at 4ºC. Transfer efficiency was assessed by staining membranes briefly with Ponceau S solution and then membranes were blocked with blocking buffer (WBAVDFL01, Millipore) diluted 1x in TBS (20 mM Tris, 150 mM NaCl, pH 7.6) for 1h followed by primary antibody (1:2000 antihuman CXCR4, ab2074; 1:100 anti-human CXCR2, ab21641; 1:1000 phospho-CXCR4, ab74012; 1:1000 phospho-CXCR2, ab61100; 1:20000 anti-GAPDH, ab8245; all antibodies were from Abcam) diluted in blocking buffer (WBAVDFL01, Millipore) overnight at 4°C with slow shaking. Membranes were then incubated with Alexa fluor 790 donkey anti-rabbit