Enhanced chemoluminescence substrate for horseradish peroxidase

ECs were harvested in electrophoresis buffer (125 mM Tris, pH 6.8, 12.5% glycerol, 2% SDS, and trace bromophenol blue), and proteins separated by SDS-polyacrylamide gel electrophoresis. After electrophoretic transfer to nitrocellulose membranes, membranes were blocked with PBS and nonfat milk (5%), and then incubated overnight at 4°C with primary antibodies against cyclin D1 (1:200), cyclin E (1:100), cyclin A (1:500), p27 (1:250), p21 (1:250), SGLT1 (1:1,500), SGLT2 (1:100), phospho-retinoblastoma protein (1:100), or β-actin (1:200). Membranes were then washed extensively in PBS and incubated with appropriate horseradish peroxidase-labeled secondary antibodies for 60 min at room temperature. Afterward, membranes were incubated with enhanced chemoluminescence substrate for horse radish peroxidase (GE Healthcare, Chicago, IL) and the signal intensity detected by X-ray film exposure. Blots were then stripped of antibodies by incubating membranes at 50°C for 30 min with a stripping buffer (10% SDS and 100 mM mercaptoethanol, in 62.5 mM Tris buffer, pH 6.8) before probing with other primary antibodies. Protein expression was determined by densitometry and normalized with respect to β-actin (Peyton et al., 2012 (link)).