Cell culture, transfection and co-immunoprecipitation (Co-IP)

HEK293T cells were cultured in Dulbecco's modified Eagle's medium (high glucose) supplemented with 10% fetal bovine serum in a 37°C incubator with 5% CO₂ and transfected using the Lipofectamine 2000 transfection reagent according to the manufacturer’s protocol. H9C2 cells were purchased from cell bank of our institute, cultured in the same conditions with HEK293T but transfected using Lipofectamine 3000 transfection reagent. Twenty-four hours after transfection, the cells were washed with 5 ml of ice-cold phosphate-buffered saline (PBS) twice, and lysed with 1 ml of ice-cold lysis buffer [50 mM Tris–HCl (pH 7.5), 150 mM NaCl, 5 mM ethylenediaminetetraacetic acid, 1% Triton X-100] supplemented with a protease inhibitor cocktail. The supernatant was collected using centrifugation at 12 000 × g for 30 min. Whole cell lysates were incubated with the anti-FLAG antibodies with agitation overnight, and then the mixture was incubated with Dynabeads protein G for 3 h. Recovered immune complexes were washed three times with ice-cold PBS plus 0.05% Tween-20 (PBST) buffer (137 mM NaCl, 2.7 mM KCl, 10 mM Na₂HPO₄, 2 mM KH₂PO₄ and 0.5‰ Triton X-100). All procedures are performed at 4°C. Proteins were eluted from the beads in 2 × protein loading buffer (100 mM Tris–HCl, 4% sodium dodecyl sulphate, 0.2% bromophenol blue, 20% glycerol and 200 mM DTT) and then subjected to western blotting.