Isolation of total protein extracts and cytosolic and nuclear protein fractions from AC16 cardiac cells or frozen tissue slides was performed as previously described.45 (link) For immunoblotting, protein fractions were resolved by 10% SDS–PAGE and transferred to polyvinylidene difluoride membranes. Proteins were identified by using several antibodies and a chemiluminescence kit (PerkinElmer, Waltham, MA, USA).
For coimmunoprecipitation studies, FOS antibody was first bound to Protein A/G Plus agarose beads by covalent crosslinking with dimethyl pimelimidate and following a standard method. Next, cell nuclear extracts (20 μg) were diluted with dilution buffer (10 mmol/L PBS, 50 mmol/L KCl, 0.05 mmol/L EDTA, 2.5 mmol/L MgCl2, 8.5% glycerol, 1 mmol/L dithiothreitol, 0.1% Triton X-100, BSA 2%, and 1 mg/mL nonfat milk) and incubated on a rocker platform for 18 h at 4 °C with 100 μL Protein A/G Plus agarose beads containing 2 μg of bound FOS antibody. The agarose beads were centrifuged and washed with PBS. After centrifugation, the protein was eluted, and the supernatant was subjected to electrophoresis and immunoblotting with the anti-acetylated-lysine antibody.
For coimmunoprecipitation studies, FOS antibody was first bound to Protein A/G Plus agarose beads by covalent crosslinking with dimethyl pimelimidate and following a standard method. Next, cell nuclear extracts (20 μg) were diluted with dilution buffer (10 mmol/L PBS, 50 mmol/L KCl, 0.05 mmol/L EDTA, 2.5 mmol/L MgCl2, 8.5% glycerol, 1 mmol/L dithiothreitol, 0.1% Triton X-100, BSA 2%, and 1 mg/mL nonfat milk) and incubated on a rocker platform for 18 h at 4 °C with 100 μL Protein A/G Plus agarose beads containing 2 μg of bound FOS antibody. The agarose beads were centrifuged and washed with PBS. After centrifugation, the protein was eluted, and the supernatant was subjected to electrophoresis and immunoblotting with the anti-acetylated-lysine antibody.
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