Cell Lysate Preparation and Western Blotting

To prepanre cell lysates, RIPA Lysis and Extraction Buffer (Invitrogen; ThermoFisher Scientific, Inc., MA, USA) which contained 10% protease inhibitor (Thermo Scientific, USA) were used to extract cell lysates by incubated on ice for 30 min, and then centrifuged at 14000 x g for 15 min at 4 °C. The protein concentrations were determined by BCA kit (Thermo Scientific, USA). For western blotting, we used the protocol which was performed previously [31 (link)]. In brief, we first mixed supernatants with 4x SDS-PAGE sample loading buffer, and they were denatured at 95 °C for 10 min. Second, the proteins were separated by SDS-PAGE gel, transferred to a polyvinylidene difluoride membranes, incubated with specific primary antibodies at 4 °C overnights, and detected with horseradish peroxidase (HRP)-conjugated secondary antibodies by using a VersaDoc Image System (BioRad, Hercules, CA, USA). For immunoprecipitation, the lysate was treated using the Dynabeads™ Protein G Immunoprecipitation Kit (Invitrogen; ThermoFisher Scientific, Inc., MA, USA) according to the protocol. The final precipitated proteins were analyzed via western blotting with the corresponding antibodies.