4.18. Western Blot Analysis

Cells were lysed with RIPA buffer containing EDTA-free Complete Protease Inhibitor Cocktail as per the manufacturer’s instructions (Roche Diagnostics, Indianapolis, IN, USA), and 1% phosphatase inhibitor 3 as previously described Sigma [25,46]. The protein concentration was determined by the DC Protein Assay (Bio-Rad, Hercules, CA, USA) and quantified using BioTek Synergy H4 (BioTek, Winooski, VT, USA)). For Western blot analysis, proteins were separated on TGX Stain-Free^TM gels (Bio-Rad), along with Precision Plus Protein^TM All-Blue Standards (Bio-Rad), and transferred to an LF PVDF membrane using the Trans-Blot Turbo Transfer System (Bio-Rad). Membranes were blocked for 1 h at room temperature in 5% non-fat dry milk in TBS-T (137 mM NaCl, 20 mM Tris, and 0.02% Tween 20). Membranes were washed following antibody incubation with TBS-T for a total of three 10-min washes. The correct molecular weight for each protein was confirmed by the Precision Plus All Blue Standard (Bio-Rad). The following antibodies were diluted in either 5% non-fat dry milk or 5% BSA in TBS-T, as per the manufacturer’s instructions, and used for detection: Rabbit anti-phospho-CHK1 serine 345 [pCHK1-Ser345] (56 kDa, cat# 2348, Cell Signaling Technology, Danvers, MA, USA); mouse IgG1 anti-CHK1 [2G1D5] (56 kDa, cat# 2360, Cell Signaling Technology); mouse anti-c-Myc [9E10] (57–65 kDa, cat# MA1-980, ThermoFisher); rabbit anti-BRD2 [D89B4] (110 kDa, cat# 5848, Cell Signaling Technology); mouse anti-BRD3 [2088C3a] (80 kDa, cat# sc-81202, Santa Cruz Biotechnology, Dallas, TX, USA); rabbit anti-BRD4 [E2A7X] (200 kDa, cat# 13440, Cell Signaling Technology); rabbit anti-RAD21 (130 kDa, cat# 4321, Cell Signaling Technology); rabbit anti-phospho-H2A.X serine 139 [p-H2AX-Ser139] (15 kDa, cat# 2577, Cell Signaling Technology); rabbit anti-H2A.X (15 kDa, cat# 2595, Cell Signaling Technology); rabbit anti-cleaved PARP [D64E10] (89 kDa, cat# 5625, Cell Signaling Technology); rabbit anti-PARP (89 and 116 kDa, cat# 9542, Cell Signaling Technology); and rabbit anti-vinculin [E1E9V] (124 kDa, cat# 13901, Cell Signaling Technology). Blots were then incubated with the appropriate horseradish peroxidase-conjugated secondary antibody, diluted 1:5000 in 5% non-fat dry milk for 1 h at room temperature (Anti-mouse IgG HRP-conjugate, cat# W4021, Promega; Anti-rabbit IgG HRP-conjugate, cat# W4011, Promega). Membranes were again washed following secondary antibody incubation with TBS-T for a total of three 10-min washes. Proteins were detected using the SuperSignal Western Chemiluminescent Substrate (Thermo Scientific, Grand Island, NY, USA) and imaged using the Bio-Rad ChemiDoc Imaging System (Bio-Rad). The quantification of protein expression was done using the Image Lab software (Bio-Rad) and proteins of interest were normalized to total protein, as quantitated from the corresponding blot, and expressed relative to control cells (NHOSTs), media- or vehicle-treated control cells, or non-targeting control siRNA. For phosphorylated CHK1 and H2AX, protein levels were also quantitated and normalized to their total protein levels, respectively.