Immunoprecipitation and Western Blotting Protocol

For immunoprecipitation, whole-cell extracts were lysed using IP Lysis Buffer (Pierce, 87785) supplemented with a protease inhibitor “cocktail” (Sigma, P8340). Cell lysates were centrifuged for 15 min at 12,000 g under 4°C, supernatants were collected and incubated with protein A/G magnetic beads (MedChemExpress, MCE, HY-K0202) together with specific antibodies. After overnight incubation, protein A/G magnetic beads were washed three times with IP wash buffer. Immunoprecipitates were eluted by SDS–PAGE loading buffer or Elution buffer (Pierce, 88848). For immunoblot analysis, cells were washed two times with cold PBS and lysed with cell lysis buffer (Cell Signaling Technology, 9803) supplemented with a protease inhibitor “cocktail” (Sigma, P8340). Protein concentrations of the extracts were measured by BCA assay (Pierce, 23235). Equal amounts of the extracts were loaded and subjected to SDS-PAGE, transferred onto polyvinylidene fluoride membrane (Millipore, IPVH00010), and then blotted as described previously (28 (link)). The specific protein bands were visualized by using a Pierce chemiluminescence ECL kit (Thermo Fisher Scientific, Waltham, MA, United States).