Ab181593

Total proteins were extracted from all cells and BCA method was used to detect protein concentration. The 10% SDS-PAGE was prepared and the gel electrophoresis board was put into the electrophoresis tank. Then, protein samples were subjected to SDS-PAGE electrophoresis. After electrophoresis, the protein samples were transferred to nitrocellulose membrane, which was transferred at the current of 95 mA for 3 h. After incubating in a blocking fluid containing skimmed milk powder for 4 h, membrane was incubated with ANXA7 (ab197586; Abcam; dilution, 1:2000), cell nuclear antigen (PCNA) (ab92552; Abcam; dilution, 1:2000), KI67 (ab16667; Abcam; dilution, 1:1000), cyclin dependent kinase 1 (CDK1) (ab32094; Abcam; dilution, 1:2000), cyclinB1 (ab181593; Abcam; dilution, 1:2000) and CDC5L (ab129114; Abcam; dilution, 1:2000) overnight at 4 °C. The next day, membrane was washed with TBST buffer containing 0.1% Tween20 for four times and incubated with goat anti-rabbit IgG-HRP second antibody (ab6721; Abcam; dilution, 1:2000) at room temperature for 1 h. After the washing of TBS, membrane was disposed with ECL and photographed with a Vilber Lourmat in the darkroom. Image J analysis system was used to analyze the gray values of each band.

RIPA lysis buffer was used to extract protein, and the protein concentration was quantified using the BCA protein concentration determination kit. The proteins (50 μg per sample) were resolved by electrophoresis under 100 V constant pressure and subsequently electro-transferred onto a PVDF transfer membrane. Then, the membrane was incubated in 5% bovine serum albumin (BSA) in TBST buffer at room temperature for 2 h, followed by incubation at 4 °C overnight with the primary antibodies as follows: anti-β-actin (1:5000; ab179467, Abcam), anti-CDC2 (1:1000; ab201008, Abcam), anti-CDK2 (1:1000; ab32147, Abcam), anti-Cyclin B1 (1:2000; ab181593, Abcam), anti-P21 (1:1000; ab188224, Abcam), anti-P27 (1:2000; ab193379, Abcam), anti-Wnt1 (1:500; 27935-1-AP, Proteintech), anti-β-catenin (1:500; ab68183, Abcam), anti-PRC1 (1:5000; ab51248, Abcam), and anti-NUF2 (1:1000; ab230313, Abcam). All antibodies were diluted with a TBST buffer containing 5% BSA. Next, the membranes were washed three times with TBST and incubated with secondary antibodies for 1 h. The membranes were then incubated with ECL chemiluminescence, and the bands were observed using a UVP chemiluminescence imaging system. We used β-actin protein as the internal reference protein to calculate the expression of the protein to be tested.

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.

Rabbit polyclonal anti‐gamma H2AX antibody (ab2893), rabbit monoclonal anti‐Cyclin B1 antibody (ab181593), mouse monoclonal anti‐CDK1 antibody (ab18), and sheep polyclonal anti‐BUBR1 antibody (ab28193) were from Abcam (Cambridge, UK). Rabbit polyclonal anti‐MAD2 antibody (10337‐1‐AP) and CoraLite594‐conjugated donkey anti‐Rabbit IgG (H + L) (SA00013‐8) were from Proteintech (Wuhan, China). Alexa Fluor 594 goat anti‐rabbit antibody, Alexa Fluor 488, and 594 goat anti‐sheep antibody were from Invitrogen (Carlsbad, CA). Rabbit polyclonal anti‐centromere (15‐234‐0001) was from Antibodies Incorporated (Shenzhen, China). Horseradish peroxidase‐conjugated goat anti‐rabbit/mouse IgG antibodies were from Beyotime (Nantong, China). Mouse monoclonal anti‐α‐tubulin‐FITC antibody (F2168), Hoechst 33342 (B2261) and all other unstated chemicals were from Sigma (St. Louis, MO).

Western blot analysis was performed as previously described [24 (link)]. Briefly, total protein was isolated from cells using the RIPA lysis buffer (Beyotime). After measuring the protein concentration using a BCA kit (Beyotime), a total of 40 μg protein was separated by 8% or 15% sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels and transferred onto the polyvinylidene fluoride membranes (Merck Millipore, Darmstadt, Germany). Membranes were subsequently blocked with 5% skim milk at room temperature for 2 h, and incubated at 4°C overnight with primary antibodies: anti-CCT3 (Abcam, Cambridge, UK, ab225878, 1:2,000), anti-cyclin B1 (Abcam, ab181593, 1:2,000), anti-cyclin dependent kinase 1 (CDK1, Abcam, ab133327, 1:10,000), anti-Cleaved Caspase-3 (Abcam, ab2302, 1:500), anti-STAT3 (Abcam, ab119352, 1:5,000), anti-p-STAT3 (Abcam, ab76315, 1:5,000), anti-JAK2 (Cell Signaling Technology, Inc., MA, USA, #3230, 1:1,000), anti-p-JAK2 (Cell Signaling Technology, #4406, 1:1,000), and anti-GAPDH (Beyotime, AG019, 1:1,000). After incubation with corresponding horseradish peroxidase-conjugated secondary antibodies (Beyotime, A0208, A0216, 1:5,000) at room temperature for 2 h, membranes were visualized under a ChemiDoc XRS+ system (Bio-Rad). The protein bands were quantified using the Image J software (National Institutes of Health, Bethesda, USA).