Western Blot Analysis of Protein Extracts

For all Western blot analyses, 1 × 10⁷ cells were collected, washed with PBS supplemented with protease inhibitor cocktail (11836153001, Sigma-Aldrich) and 1 mM PMSF (Sigma-Aldrich), and then lysed with Laemmli lysis buffer as previously described (46 (link)). Equal amounts of the protein extracts (~10 μg) were electrophoresed on NuPAGE 4–12% Bis-Tris gradient gels (Novex, Carlsbad, CA) and then electro-blotted onto Immobilon PVDF membranes (EMD Millipore, Billerica, MA). Membranes were probed with the following Abs and dilutions: GABPα (1:1000; SC-22810), PU.1 (1:1000; SC-352), C/EBPε (1:1000; SC-158), and β-actin (1:1000) (all from Santa Cruz Biotechnology, Dallas, TX), LBR from guinea pig serum [kindly provided by Prof. Harald Herrmann, German Cancer Research Institute, Heidelberg, Germany and previously described (20 (link))], or tubulin (1:1500; Sigma-Aldrich). HRP-conjugated secondary Ab (1:2000, Sigma-Aldrich) was used against primary Abs, and chemiluminescence was detected using Immobilon Western HRP substrate solution (EMD Millipore). Images were captured from exposed and processed film or with the ChemiDocMP Imager using Image Lab Software (BioRad Laboratories).