Immunoprecipitation and immunoblotting analysis

Protein samples were extracted from transfected cells using lysis buffer (50 mM Tris-HCl, pH 7.5, 0.01% SDS, 1% Triton X-100, 150 mM NaCl, 1 mM dithiothreitol, 0.5 mM EDTA, 100 μM PMSF, 100 μM leupeptin, 1 μM aprotinin, 100 μM sodium orthovanadate, 100 μM sodium pyrophosphate, and 1 mM sodium fluoride). Cellular extracts were then centrifuged at 12,000 × g at 4 °C, and the supernatants were collected. For immunoprecipitation, 1 mg protein lysates were incubated with 10 μg antibodies overnight at 4 °C. Next, protein G-tagged beads were added to the lysates for another 3 h. Lysis buffer was used to wash the immunocomplexes 3 times. Immunoprecipitation products were analyzed using an immunoblotting assay against the indicated antibodies as previously described⁵¹. Briefly, immunoprecipitation products were separated on SDS-polyacrylamide gels and transferred onto polyvinylidene difluoride (PVDF) membranes. After blocking with 5% skim powdered milk for 2 h, the membranes were incubated with primary antibodies overnight at 4 °C and with HRP-conjugated secondary antibodies for 2 h at room temperature. The blots were visualized by ECL chemiluminescent reagent. Antibodies was diluted at 1:1000 for immunoprecipitation and 1:50 for immunoblotting analysis. The antibodies used in this study are listed in the Materials section.