Horseradish peroxidase conjugated anti rabbit igg antibody

For coimmunoprecipitation experiments, cells were harvested in PBS, centrifuged at 800 g for 10 min, and lysed in 125 mmol/L NaCl, 50 mmol/L Hepes, pH 7.4 (supplemented with 1% Triton X-100 or CHAPS and Complete protease inhibitors) for 30 min at 4 °C. After centrifugation at 16000 g for 15 min, cleared cell extracts were incubated overnight at 4 °C with protein A/G-agarose (Santa Cruz Biotechnology, Santa Cruz, CA, United States) and anti-PSN1 or anti-rabbit IgG beads. Immunoprecipitated proteins were resolved on 12% SDS-polyacrylamide gels and transferred to PVDF membranes (Bio-Rad, Richmond, CA, United States). The membranes were blocked for 1 h in PBS containing 0.1% Tween 20 (PBS-T) and 5% non-fat dry milk (Sigma) and reacted with antibodies against PSN1, each diluted 1:1000. The membranes were washed with PBS-T, then incubated for 1 h at room temperature with horseradish peroxidase-conjugated anti-rabbit IgG antibody (Sigma), diluted 1:5000, and developed with westernsure ECL substrate (LI-COR Biosciences). Immunoreactive bands were identified by co-migration of prestained protein size markers (Fermentas, Glen Burnie, MD, United States). To confirm equivalent protein loading and transfer, the blots were stripped and re-probed for GAPDH (Santa Cruz Biotechnology).