Ab1930

Porcine EPCs (approximately 40% confluent in a T25 flask) were trypsinized, centrifuged (350 × g, 3.5 min) and then extracted in 400 μl RIPA lysis buffer with protease inhibitors (Sigma, cat. no. P8340) and 3X protein sample buffer. Proteins in cell extracts (30 μl per lane) were separated on 8% SDS-PAGE gels under non-reducing conditions, and transferred to PVDF membranes for 2 h with methanol on ice. Blots were then blocked with 2% milk on a shaker for 1 h at room temperature, incubated with milk containing polyclonal rabbit αv antibody (EMD Millipore, AB1930, Billerica, MA) at a 1:1000 dilution, mouse GAPDH antibody (Abcam, AB9483, Cambridge, MA) at a 1:10000 dilution, and mouse alpha tubulin antibody (Enzo Life Sciences, cat. no. IMG-80196, Farmingdale, NY) at a 1:10000 dilution overnight at 4°C with gentle mixing, and then incubated in the corresponding rabbit or mouse HRP-conjugated secondary antibodies (Dako, Carpinteria, CA) at a 1:5000 dilution in milk for 1 h at room temperature. Immunoreactive proteins were detected using Immobilon Western Chemiluminescent HRP Substrate (2 ml per blot) (EMD Millipore, lit. no. P36599A Billerica, MA) and blots were developed and scanned by a HP Scanjet G3010 Photo Scanner (Hewlett-Packard, Palo Alto, CA).