Ab133327

Total protein was harvested from cells using RIPA lysis buffer (Beyotime, China). Protein samples were separated by sodium dodecyl-sulfate polyacrylamide (SDS-PAGE) gel and transferred to polyvinylidene fluoride (PVDF) membranes. Blots were washed and incubated with antibodies. The protein bands were then visualized with an ECL detection system. The primary and secondary antibodies utilized in this study were listed as follows: anti-GAPDH (60004-1-Ig, Proteintech, USA), anti-HNRNPAB Abcam, USA), anti-CDK1 (ab133327, Abcam, USA), anti-Cyclin B1 (ab32053, Abcam, USA), anti-CDC25A (sc-7389, Santa Cruz, USA), anti-CDC25C (ab32444, Abcam, USA), anti-ER (ab75635, Abcam, USA), anti-HER2 (ab134182, Abcam, USA), HRP-conjugated goat anti-mouse IgG (SA00001-1, Proteintech, USA), and HRP-conjugated goat anti-rabbit IgG (SA00001-2, Proteintech, USA).

Antibodies against CDK1 (ab133327), Cyclin B1 (ab32053) and CDC25C (ab32444), Caspase-3 (ab32351), Bcl2 (ab182858), LC3B (ab192890), SQSTM1/P62 (ab109012), and γH2AX (ab81299) were obtained from Abcam (Cambridge, MA, USA). Antibodies against NEK2 (66632-1-lg), TRIM21(12108-1-AP), α-tubulin (11224-1-AP), GAPDH (60004-1-Ig), Bax (60267-1-Ig), Alexa Fluor 594-conjugated donkey anti-rabbit second antibody (SA00013-4), and Alexa Fluor 488-conjugated donkey anti-mouse second antibody (SA00013-1) were obtained from Proteintech (Wuhan, China). An antibody against NEK2 (sc55601) was got from Santa Cruz Biotechnology, Inc (St Louis, MO, USA). The autophagy activator Rapamycin (RAPA, HY-10,219), Cycloheximide (CHX, HY-12,320), and MG132(HY-13,259) were purchased from MedChemExpress (MCE, USA).

Total protein was extracted from the tissue samples and cells, and the protein concentrations were quantified using a BCA kit (Yeasen, Shanghai, China). Equivalent proteins were separated by SDS-PAGE and transferred to PVDF membranes (Millipore, Billerica, MA, USA). Then, the membranes were blocked and incubated with primary and secondary antibodies. We used ECL chemiluminescence to detect protein signals. GAPDH was used as the internal reference protein. Antibodies against the following proteins were used: GAPDH (60004-1-Ig, Proteintech), FEN1 (ab133311, Abcam), Cdc25C (ab32444, Abcam), CDK1 (ab133327, Abcam) and Cyclin B1 (ab32053, Abcam).

OSW-1 (C47H68O15, purity ≥95%) was purchased from Cayman Chemical Company (Ann Arbor, Michigan, United States), dissolved in dimethyl-sulfoxide DMSO (Sigma-Aldrich, St. Louis, MO, United States) then stored in the dark at − 20°C. OSW-1 was diluted with cell culture medium to the required concentrations, with DMSO concentration <0.1%, to avoid side effects. Dulbecco’s modified Eagle medium (DMEM) and fetal bovine serum (FBS) were purchased from Thermo Fisher Scientific (Gibco, United States). Cell counting Kit-8 (CCK-8), RNase, penicillin/streptomycin, propidium iodide, Annexin V-FITC Apoptosis Detection Kit, RIPA lysate, protease inhibitor, protein phosphatase inhibitor, BCA protein assay kit, and electrochemiluminescence (ECL) kit were purchased from Beyotime Biotechnology Company (Shanghai, China). 740Y-P was purchased from TargetMol (Shanghai, China). Primary antibodies against P21 (ab109520), cyclin B1 (ab32053), CDK1 (ab133327), PARP-1 (ab191217), cleaved-PARP-1 (ab32064), cleaved caspase-3 (ab214430), beta Actin (ab8226), PI3K(ab191606), p-PI3K (ab278545), AKT1 (ab183556), and p-AKT1 (ab81283) were purchased from Abcam (Cambridge, MA, United Kingdom). Primary antibodies against cleaved caspase-9 (#9502) and secondary antibodies (goat anti-mouse IgG and goat anti-rabbit IgG, #98164 and #91196) were purchased from Cell Signaling Technology (Beverly, MA, United States).

Total protein was extracted from cells using the radioimmunoprecipitation assay lysis buffer containing phenylmethylsulfonyl fluoride at a final concentration of 1 mmol/L (Beyotime Co, Shanghai, China). Western blotting was performed using antibodies against CDKN1C/p57 (Cell Signaling Technology [CST]; #2557), CDK1 (Abcam; ab133327), CDK2 (CST; #2546), CDK6 (CST; #3136), CCNA2 (CST; #4656), and CCNB1 (CST; #12231), as previously described (Song et al., 2019 (link)). β-Actin (Abcam; ab8226) was used as an endogenous reference.

For cellular experiments, total proteins were extracted using RIPA lysis buffer supplemented with protease inhibitor cocktails. Then, the samples were separated by SDS-PAGE and transferred to polyvinylidene difluoride (PVDF) membranes, as described before. Subsequently, the membranes were blocked with 5% nonfat milk in TBST and immunoblotted with anti-cleaved caspase3 (#9661S, 1:1000, Cell Signaling Technology, Danvers, MA, USA), anti-p21 (ab109199, 1:1000, Abcam), anti-CCNB1 (ab181593, 1:1000, Abcam), anti-CDK1 (ab133327, 1:1000, Abcam), anti-RB (ab181616, 1:1000, Abcam), anti-p-RB (#8516S, 1:1000, Cell Signaling Technology) primary antibodies, and GAPDH (AC033, 1:30000, ABclonal, Wuhan, China) overnight at 4 °C. Then, the PVDF membranes were washed and incubated with HRP-conjugated anti-rabbit IgG secondary antibody (#7076, 1:2000, Cell Signaling Technology) or HRP-conjugated anti-mouse IgG secondary antibody (#7074, 1:2000, Cell Signaling Technology). Protein expression level was detected using an enhanced chemiluminescence (ECL) kit and photographed under the automatic chemiluminescence image analyzer (#5200, Tanon, Shanghai, China). Relative quantification was analyzed based on Image J 1.53v software.