Supersignal west dura extended duration substrate system

Equal amounts of protein were used as total extracts, followed by SDS-PAGE, Western blot analysis with the indicated antibodies and imaging using the SuperSignal™ West Dura Extended Duration Substrate System (Thermo Fisher Scientific, San Jose, CA, USA) and Gel Doc XR+ system (Bio-Rad, Hercules, CA, USA) or G:BOX Chemi XX6 gel doc systems (Syngene, Cambridge, UK). Antibodies against STAT3 (sc-7179), STAT5 (sc-835), AURKA (sc-25425), AURKB (sc-25426), phospho (p)-histone H3^S10 (sc-8659-R), and p-histone H2A.X^S139 (γ-H2A.X; sc-517348) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Antibodies against p-STAT3^Y705 (#9131S), p-STAT5^Y694 (#9359S), and caspase 3 (#9665) were obtained from Cell Signaling Technology (Danvers, MA, USA). Cropped gels retain important bands, but whole gel images are available in Supplementary Fig. 7.

Cells were treated with vehicle or THZ-P1-2 (1.6, 3.2, or 6.4 μM) for 24 h and submitted to total protein extraction using a buffer containing 100 mM Tris (pH 7.6), 1% Triton X-100, 2 mM PMSF, 10 mM Na₃VO₄, 100 mM NaF, 10 mM Na₄P₂O₇, and 4 mM EDTA. Equal amounts of protein (30 μg) from the samples were subsequently subjected to SDS-PAGE in an electrophoresis device, followed by electrotransfer of the proteins to nitrocellulose membranes. The membranes were blocked with 5% non-fat dry milk and incubated with specific primary antibodies diluted in blocking buffer, followed by secondary antibodies conjugated to horseradish peroxidase (HRP). Western blot analysis was performed using a SuperSignal^TM West Dura Extended Duration substrate system (Thermo Fisher Scientific) and G: BOX Chemi XX6 gel document system (Syngene Cambridge, UK). Antibodies directed against PARP1 (#9542), γH2AX (#9718), SQSTM1/p62 (#88588), LC3B (#2775), and α-tubulin (#2144) were obtained from Cell Signaling Technology (Danvers, MA, USA). Band intensities were determined using UN-SCAN-IT gel 6.1 software (Silk Scientific; Orem, UT, USA).

Equal amounts of protein were used as total extracts, followed by SDS-PAGE, Western blot analysis with the indicated antibodies. For imaging the SuperSignal^TM West Dura Extended Duration Substrate System (Thermo Fisher Scientific, USA) and Gel Doc XR + system (Bio-Rad, Hercules, CA, USA) were used. Antibodies against CDKN1A (sc-71811), MLL5 (sc-377182), OP18 (sc-55531), and α-tubulin (sc-5286) were obtained from Santa Cruz Biotechnology (San Jose, CA). Antibodies against H3K4me3 (#9727) and fibrillarin (#2639) were obtained from Cell Signaling Technology (Danvers, USA). All membranes were incubated with a primary antibody following manufacturer’s instructions (see Supplementary Table S1). Band intensities of cropped gels, which retained important bands were measured by the UN-SCAN-IT gel 6.1 software (Silk Scientific; USA).

Total protein extraction was performed using a buffer containing 100 mM Tris (pH 7.6), 1% Triton X-100, 150 mM NaCl, 2 mM PMSF, 10 mM Na₃VO₄, 100 mM NaF, 10 mM Na₄P₂O₇, and 4 mM EDTA. Equal amounts of protein were used from total extracts followed by SDS-PAGE, and Western blot analysis with the antibodies indicated, as previously described (29 (link)) Antibodies against total and cleaved PARP1 (#9542), LC3BI/II (#2775), antibody against γ-H2AX (#9718), anti-rabbit HPR (#7074), procaspase 3 (#9665), cleaved-caspase 3 (#9665) and α-tubulin (#2144) were obtained from Cell Signaling Technology (Danvers, MA, USA). Antibody binding was revealed using a SuperSignalTM West Dura Extended Duration substrate system (Thermo Fisher Scientific) and a G: BOX Chemi XX6 gel document system (Syngene, Cambridge, UK). All the experiments were repeated at least 3 times.