Protein g or a agarose

Subcellular fractionation was performed as described previously (2 (link)). Briefly, cells were washed with ice-cold phosphate-buffered saline (PBS) and centrifuged at 2500 x g at 4°C. Pellets were subsequently resuspended in buffer A (10 mM HEPES, pH 7.9, 10 mM KCL, 0.1 mM EDTA, 0.1 mM EGTA, 1 mM DTT, protease inhibitors) for 30 minutes and consequently centrifuged for 20 minutes at 5000 x g. Supernatants were transferred to new tubes and centrifuged for 20 minutes at 12000 x g. Pellets were resuspended in buffer C (20 mM HEPES, pH 7.9, 0.4 mM NaCL, 1 mM EDTA, 1 mM EGTA, 1 mM DTT, protease inhibitors) and incubated on ice for additional 30 minutes. The final centrifugation step was carried out at 12000 x g for 10 minutes.
For Coimmunoprecipitation (Co-IP), cells were lysed with lysis buffer (50 mM HEPES; pH 7.5–7.9, 150 mM NaCl, 1 mM EGTA, 10% Glycerin, 1% Triton X-100, 100 mM NaF, 10 mM Na₄P₂O₇ × 10 H₂0) containing protease inhibitors. 500 μg of the total lysates or 100 μg of nuclear lysates were immunoprecipitated with the indicated antibodies and protein G- or A-agarose (Roche diagnostics, Mannheim, Germany).
For isolation of proteins from murine tissues, the tissues were homogenized using a mortar and subsequently mixed with lysis buffer. After sonification, cells were centrifuged at 12000 x g for 10 minutes and subjected to immunoblotting.