Protein a g agarose beads

Proteins were extracted from HK2 cells with a lysis buffer (50 mM Tris–HCl pH 7.5, 10 mM NaF, 0.1% deoxycholate, 10 mM β-glycerophosphate, 1% NP-40, 1 mM Vanadate, 1 mM PMSF, protease inhibitor, and Phosphatase Inhibitor) and were centrifuged at a speed of 12,000×g for 20 min. The supernatants were quantified by BCA Protein Assay Kit (Thermo Scientific) and approximately 1.2 mg protein were subjected to immunoprecipitation with ANTI-FLAG M2 antibody (Sigma–Aldrich). The mixture was incubated at 4 °C overnight, then the protein A/G agarose beads (Smart-Lifesciences) were added. The mixture was incubated at 4 °C for additional 4 h. After that, the immunocomplexes were centrifugated and washed with the lysis buffer for four times. Finally, these immunocomplexes were subjected to Western blotting analysis.