Sc 490

Cell extracts were prepared using RIPA lysis buffer (150 mM sodium chloride, 1% NP-40, 0.1% SDS, 50 mM Tris, pH 7.4) containing 1 mM β-glycerophosphate, 2.5 mM sodium pyrophosphate, 1 mM sodium fluoride, 1 mM sodium orthovanadate, and protease inhibitor (Roche, Basel, Switzerland). Protein concentration was quantified using Bradford assay reagent (Bio-Rad) according to manufacturer instructions. Proteins were resolved by SDS-PAGE and then transferred to a polyvinylidene fluoride membrane (Pall Corporation, Port Washington, NY, USA). Membranes were blocked with 5% non-fat milk and incubated with the following antibodies at the indicated dilutions: anti-p21 (1:500; sc-397), anti-IκBα (1:500; sc-371), anti-p53 (1:500; sc-126, all from Santa Cruz Biotechnology), anti-p-p53 (1:500; 9286, Cell Signaling Technology), anti-ID1 (1:2,000; BCH-1-195-14, Biocheck, Foster City, CA, USA), anti-ID2 (1:500, sc-489, Santa Cruz Biotechnology), anti-ID3 (1:500, sc-490, Santa Cruz Biotechnology), anti-ID4 (1:200; ab49261, Abcam), and anti-β-actin (1:10,000; A5316, Sigma-Aldrich). Membranes were then incubated with a horseradish peroxidase-conjugated anti-IgG secondary antibody (Pierce Biotechnology, Rockford, IL, USA) and visualized using SuperSignal West Pico Chemiluminescent Substrate (Pierce Biotechnology).

Western blot analysis of C2C12 myoblasts was performed as described (Micheli et al., 2011 (link)). Briefly, cells were lysed by sonication in buffer containing 50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1 mM EDTA, 0.2% Nonidet P-40, with protease inhibitors 1 mM Na₃VO₄, 10 mM 2-glycerophosphate, 10 mM NaF, 5 mM ATP, 5 mM MgCl₂. Proteins were electrophoretically separated by SDS-PAGE and transferred to nitrocellulose filters. Immunoblots were performed hybridizing filters to a rabbit polyclonal antibody against Id3 (Santa Cruz Biotechnology; Sc-490, 1:300); detection of the second antibody (goat anti-rabbit horseradish peroxidase-conjugated antibody; Jackson ImmunoResearch) was performed by chemiluminescent assay.