Transfected HEK293T cells were harvested and lysed in IP lysis buffer (250 μl) with rotation at 4°C for 1 h and the lysates were clarified by centrifugation (20,000 × g for 20 min at 4°C). Clarified lysates (25 μl) was mixed with 4 × lithium dodecyl sulfate buffer (LDS, ThermoFisher Scientific) containing 100 mM 1,4-dithiothreitol, samples were heated at 70°C for 10 min, and stored at −20°C or analyzed by western blot as input whole cell lysis (WCL). For the rests of the lysates, immunoprecipitation was performed by using specific antibodies, extra NaCl was added to a final concentration of 300 mM. Subsequently, the immune complexes were captured by protein G-conjugated magnetic beads (Millipore) according to the manufacturer’s instructions. After extensively washing, beads were eluted by denaturation in 2 × LDS buffer at 70°C for 10 min.
Protein samples were resolved by SDS-polyacrylamide gel electrophoresis and transferred onto a polyvinylidene difluoride (PVDF) membrane using Trans-Blot Turbo Transfer System (Bio-Rad, Hercules, CA), followed by blocking for 1 h and probing with an indicated primary antibody and an anti-Rabbit/Mouse IgG-Peroxidase antibody (Sigma-Aldrich, 1:20,000). The proteins were visualized using SuperSignal Femto Maximum Sensitivity Substrate (ThermoFisher Scientific) and ChemiDoc Imaging Systems (Bio-Rad).
Protein samples were resolved by SDS-polyacrylamide gel electrophoresis and transferred onto a polyvinylidene difluoride (PVDF) membrane using Trans-Blot Turbo Transfer System (Bio-Rad, Hercules, CA), followed by blocking for 1 h and probing with an indicated primary antibody and an anti-Rabbit/Mouse IgG-Peroxidase antibody (Sigma-Aldrich, 1:20,000). The proteins were visualized using SuperSignal Femto Maximum Sensitivity Substrate (ThermoFisher Scientific) and ChemiDoc Imaging Systems (Bio-Rad).