Epr3324

Cells were lysed in ice-cold RIPA with protease and phosphatase inhibitors (Santa Cruz, Dallas, TX). Whole-cell extracts were prepared by centrifugation (14,000 × g, 15 min) to remove insoluble components. Protein concentrations were determined by BCA assays (Thermo Scientific, Waltham, MA) and loading volumes were normalized. Proteins were then separated by 8% or 4–20% gradient SDS-PAGE (Bio-Rad, Hercules, CA) and transferred to PVDF membranes. Primary antibodies for protein detection included: phospho-H2AX (γH2AX, JBW301, Millipore, Billerica, MA), PARP1 (F-2, Santa Cruz), PAR (Trevigen, Gaithersburg, MD), Actin (C-2, Santa Cruz), XRCC1 (mouse monoclonal, Abcam, Cambridge, UK), small subunit μ-calpain (EPR3324, Abcam). Primary hybridizations were performed in Sigma casein blocking buffer at 4 °C overnight. Secondary HRP-conjugated antibodies were incubated for 1 h at room temperature, followed by detection with SuperSignal West Pico (Thermo Scientific). Bands were quantified by mean intensity using NIH ImageJ, and normalized to actin.

Cells were lysed in ice-cold RIPA with protease and phosphatase inhibitors (Santa Cruz, Dallas, TX, USA). Whole-cell extracts were prepared by centrifugation at 14 000 × g to remove insoluble components. Protein concentration was determined by BCA assay (Thermo Scientific, Waltham, MA, USA) and loading volume was normalized. Extracts were run on 8 or 4–20% (Bio-Rad, Hercules, CA, USA) gradient acrylamide SDS-PAGE gels and transferred to PVDF membrane. Primary antibodies for protein detection included: phospho-H2A.X (JBW301, Millipore, Billerica, MA, USA), PARP1 (F-2, Santa Cruz), PAR (Trevigen, Gaithersburg, MD, USA), Actin (C-2, Santa Cruz), visfatin/NAMPT (rabbit polyclonal, Abcam, Cambridge, UK), small subunit calpain (EPR3324, Abcam). Primary hybridization was carried out in Sigma casein blocking buffer at 4 ° overnight. Secondary HRP conjugated antibodies were incubated for 1 h at room temperature, followed by detection with SuperSignal West Pico (Thermo Scientific). Bands were quantified by mean intensity in ImageJ and normalized to the actin band intensity to control for loading variation.

Western blots were carried out as previously described [25 (link)]. Primary antibodies for protein detection included: GLS1 (ab93434, Abcam), PARP1 (F-2, Santa Cruz), PAR (Trevigen, Gaithersburg, MD), Actin (C-2, Santa Cruz), small subunit calpain (EPR3324, Abcam), and 53BP1 (Bethyl Laboratories).