Secondary horseradish peroxidase conjugated antibodies

Transient transfection was performed by using poly(ethylenimine) (PEI, polysciences). For immunoprecipitation, cells were washed with ice-cold phosphate buffered saline (PBS), and then lysed in NETN buffer (0.5% Nonidet P-40, 20 mM Tris [pH 8.0], 50 mM NaCl, 50 mM NaF, 100 μM Na3VO4, 1 mM dithiothreitol (DTT) and 50 μg/ml phenylmethylsulfonyl fluoride (PMSF)) with benzonase (Enzynomics, M018H) at 4°C for 40 min. Crude lysates were cleared by centrifugation at 14000 rpm at 4°C for 5 min, and supernatants were incubated with protein A-agarose-conjugated primary antibodies, FLAG-M2 affinity gel (Sigma, Cat#A2220) or c-Myc Agarose affinity gel (Sigma, Cat#7470). The immunocomplexes were washed three times with NETN buffer, and then subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Western blotting was performed using the antibodies indicated in the figure legend. Proteins were visualized using secondary horseradish peroxidase-conjugated antibodies (Enzo Life Sciences, New York, NY) and enhanced chemiluminescence reagent (Thermo Fisher Scientific). Signal was detected using an automated imaging system (ChemiDoc™; Bio-Rad Laboratories, Hercules, CA).