Anti myc antibody conjugated agarose beads

Total cell protein was extracted with RIPA buffer containing 50 mM Tris (pH 7.4), 1% Nonidet P-40, 0.25% sodium deoxycholate, 1 mM EDTA (pH 8.0), 150 mM NaCl, 1 mM NaF, 1 mM PMSF, 1 mM Na3VO4, a 1:100 dilution of phosphatase inhibitor cocktail (Cell Signaling Technology, #5870 S), and a 1:100 dilution of protease inhibitor mixture (Bimake, #B14001). Cell lysates were separated by SDS-PAGE, transferred to a polyvinylidene difluoride (PVDF) membrane (Millipore), blocked with 5% (w/v) nonfat milk, probed with the indicated primary antibodies and corresponding secondary antibodies, visualized using ECL Western blotting detection reagent (Millipore) and photographed using a Fuji Film LAS4000 mini-luminescent image analyzer. Anti-Flag antibody-conjugated agarose beads (Sigma-Aldrich, #A2220), Anti-Myc antibody-conjugated agarose beads (Sigma-Aldrich, #A7470), and anti-HA antibody-conjugated agarose beads (Sigma-Aldrich, #A2095) were used for the exogenous co-immunoprecipitation assay. Protein G Sepharose (GE HealthCare Company, #17-0618-01) was used for the endogenous co-immunoprecipitation assay. Image J software (National Institutes of Health) was used to quantify protein levels based on the band density obtained by Western blot analysis.