Mouse monoclonal antibodies to β actin

Cells were lysed in buffer with protease and phosphatase inhibitors (Roche Applied Science, Indianapolis, IN). Protein concentration was measured using the Pierce™ BCA Protein Assay Kit (Thermo Scientific, Waltham, MA), and 30 μg from each sample was electrophoresed (8 (link)). Immunoblots were incubated with polyclonal antibodies to Pim-1, PARP, Mcl-1, Bim, phospho-BAD^Ser112, BAD, Bax, Bak, Bcl-2, Bcl-xL, USP9X, phospho-STAT5^Tyr694 (Cell Signaling Technology, Danvers, MA), USP24 (Abcam, Cambridge, MA), vinculin and Trim17 (Sigma-Aldrich), SCFβ-TRCP and ARF-BP1 (Abcam) and mouse monoclonal antibodies to β-actin (Santa Cruz Biotechnology, Dallas, TX), glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (EMD Millipore) and FLAG (Sigma-Aldrich) overnight at 4°C, then with horseradish peroxidase-conjugated secondary antibodies for one hour at room temperature. Band intensities at serial time points, measured by densitometry (VisionWorks®LS, UVP, Upland, CA), were compared to pre-treatment intensity, defined as 100%. Measurements of Mcl-1 and USP9X expression in cells treated with inhibitors and combinations were repeated in at least four separate replicate experiments.