Tg2011 1

Western blot analysis of cell lysates was performed as previously described [10 (link)], using the primary antibodies: SMC6 GP (1/200; custom made), SMC5 (1/1000; A300-236A, Bethyl Laboratories, Montgomery, TX, USA), NSMCE2 (1/500; NBP1-76263, Novus Biologicals, Littleton, CO, USA), TOP2A (1/1,000; TG2011-1, TopoGEN, Buena Vista, CO, USA) and β-actin (1/5000; A1978, Sigma-Aldrich).

Protein lysates were prepared as detailed previously (Ahmed et al., 2019). Briefly, cells were resuspended in RIPA buffer containing Protease Inhibitor cocktail (Roche, Basel, Switzerland) on ice. The mixture was then centrifuged at 15 000 r.p.m. for 20 min at 4 °C. The supernatant was collected, and following protein concentration determination, lysates were separated by SDS/PAGE, transferred onto 0.2‐µm PVDF membranes (Bio‐Rad, Hercules, CA, USA) and blocked in superblock blocking buffer (Thermo Fisher Scientific, Waltham, MA, USA). Membranes were incubated with primary antibodies [α‐HMGA2 (CST 5269; 1 : 1000; RRID:AB_10694917), α‐TOP2A (TopoGEN TG2011‐1; 1 : 2000; RRID:AB_1934304), α‐TOP2B (Ab109524; 1 : 2000; RRID:AB_10859793), α‐β‐actin (Sigma A2228; 1 : 5000; RRID:AB_476697)] overnight at 4 °C, followed by incubation with secondary antibodies [Polyclonal goat anti‐mouse (Dako, P0447, Carpinteria, CA, USA; RRID:AB_2617137) and polyclonal goat anti‐rabbit (Dako, P0448; RRID:AB_2617138)] at room temperature for 1 h. Immunoreactivity was developed by chemiluminescent HRP substrate (Millipore, Singapore) in a luminescence imager (LAS4000; Fujifilm, Pittsburgh, PA, USA).