Protein a g agarose slurry

Immunoprecipitation was performed as reported by Cenni et al. [24 (link)]. Equal amounts of precleared lysates (pcl), whose protein concentration was determined by the Bradford method, were incubated overnight with rabbit anti-Nox4 (Novus Biologicals, CO, USA) and mouse anti-sc-35 (Sigma-Aldrich) (3 μg all). Then the two samples were treated with 30 μL of 50% (v/v) of protein A/G agarose slurry (GE Healthcare Bio-sciences, Uppsala, Sweden) at 4°C with gentle rocking for 1 h. Pellets were washed twice with 20 mM Tris-Cl, pH 7.0; 1% Nonidet P-40; 150 mM NaCl; 10% glycerol; 10 mM EDTA; 20 mM NaF; and 5 mM sodium pyrophosphate, once with 10 mM Tris-Cl, pH 7.4, boiled in SDS sample buffer, and centrifuged. Supernatants were loaded onto SDS-polyacrylamide gel, blotted on Immobilon-P membranes (Millipore, Waltham, MA, USA), processed by western blot with the indicated antibodies and detected by Supersignal substrate chemiluminescence detection kit (Pierce, Rockford, IL, USA). Signal quantification was obtained by chemiluminescence detection on a Kodak Image Station 440CF and the analysis with the Kodak 1D Image software.

Immunoprecipitation was performed as reported by Bertacchini et al. [31 (link)]. For preclearing procedure nuclear lysates were incubated with 2 μg anti-M2 for 1 hour (Sigma Aldrich) and then with beads for additionally 30 min, which were then removed and discarded prior to the immunoprecipitation. Precleared lysates, whose protein concentration was determined by the Bradford method, were incubated 4 hours with 3 μg of anti-Nox4 (Novus Biologicals, CO, USA). Then samples were treated with 30 μL of 50% (v/v) of protein A/G agarose slurry (GE Healthcare Biosciences, Uppsala, Sweden) at 4°C with gentle rocking for 1 h. Pellets were washed twice with 20 mM Tris-Cl, pH 7.0; 1% Nonidet P-40; 150 mM NaCl; 10% glycerol; 10 mM EDTA; 20 mM NaF; 5 mM sodium pyrophosphate, once with 10 mM Tris-Cl, pH 7.4, boiled in SDS sample buffer, and centrifuged. Supernatants were loaded onto SDS-polyacrylamide gel, blotted on Immobilon-P membranes (Millipore, Waltham, MA, USA), and processed by Western blot with the indicated antibodies.