Ab26212

Total protein in HCC cells was extracted by the RIPA kit (R0010, Beijing Solarbio Life Sciences Co., Ltd., Beijing, China). Total protein was separated by 10% sodium dodecyl sulphatepolyacrylamide gels electrophoresis (SDS-PAGE), which then was transferred onto the polyvinylidene difluoride (PVDF) membranes (Millipore Corp., USA). The membranes loaded with protein shaken for blockage 2h with 5% skim milk at room temperature. Subsequently, the PVDF membranes were incubated with anti-SSRP1 polyclonal antibody (ab26212, 1:1500 dilution, Abcam, Cambridge, UK, USA) and mouse against GAPDH antibody (ab128915; 1:1500 dilution; Abcam, Cambridge, UK, USA) overnight at 4°C, respectively. After that, we continued to incubate the PVDF membranes with horseradish peroxidase (HRP)-conjugated anti-mouse IgG secondary antibodies (ab205718; 1:5000 dilution; Abcam, Cambridge, UK, USA) at room temperature for 1h. The Enhanced Chemiluminescence Reagent (Bio-Rad Labora-tories, Hercules, CA, USA) were used to visualize the immunoreactive bands.

Cells were seeded on cell climbing pieces, followed by fixed by 4% paraformaldehyde, and washed by Phosphate‐Buffered Saline (PBS) solution for three times. Then, cells were incubated with 0.5% Triton X‐100 for 15 min, and blocked in FBS for 60 min. Primary antibodies (γH2AX, purchased from Abcam, Cambridge, MA, USA, ab81299; SSRP1, purchased from Abcam, ab26212; H2A, purchased from Abcam, ab177308; H2B, purchased from Abcam, ab1790) were used and incubated at 4°C overnight. Secondary antibodies (ThermoFisher, MA, USA) were used and incubated at room temperature for 1 h. 4′,6‐diamidino‐2‐phenylindole (DAPI) was used for cell nucleus staining. Pictures were taken by laser confocal microscopy.

Standard Western blot (WB) assays were performed. Anti‐SSRP1 antibody (ab26212), anti‐H2A antibody (ab28155) and anti‐H2B antibody (ab1790) were from Abcam, and anti‐GAPDH antibody was purchased from CST (51332) (Danvers, MA, USA), which was used as a control for whole cell lysates.