Anti cyclin b1

Whole cell proteins were harvested and the protein concentrations were measured using a BCA kit (Pierce; Thermo Fisher Scientific, Inc). And equal protein (30 μg) was separated with 10% SDS‐PAGE and transferred onto nitrocellulose membranes (EMD Millipore), subsequently, the membranes were then blocked in 5% non‐fat dry milk for 2 hours at room temperature. The membrane was incubated with primary antibodies at 4°C overnight, and the membrane was washed with TBST and incubated with anti‐HPR‐conjugated Affinipure goat anti‐rabbit/mouse lgG(H+L) (1:5000, Proteintech Group, Inc). The signals were detected using an ECL Western blotting kit. The primary antibodies used included: Anti‐B3GNT5 (1:500), anti‐GAPDH (1:3000), anti‐cyclinD1 (1:500), anti‐cyclinB1 (1:500 proteintech), anti‐EGFR (1:1000) (Proteintech Group, Inc) anti‐ERK (1:1000), anti‐p‐ERK (1:500) (Cell Signaling Technology, Inc); AKT (1:1000, Beyotime Institute of Biotechnology) and p‐AKT(Ser473) (1:500, Beyotime Institute of Biotechnology).

The tissue and cell proteins were isolated using radio immunoprecipitation assay (RIPA) lysis buffur (Epizyme Biotech) supplemented with protease inhibitor. The Bradford method (CWBIO) was used to determine the protein concentrations. Proteins were electrophoresed on Sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE, CWBIO), then transferred to polyvinylidene fluoride (PVDF) membranes, blocked, and incubated with primary anti-FANCE (Biobryt, 1:10000), anti-FANCD2 (1: 5000; Novus Biologicals), anti-GAPDH (1:10000, bioworld), anti-β-tubulin (1:10000, proteintech), anti-RAD51(1:5000, Abcam), anti-γH2AX (1:10000, Abcam), anti-cyclin dependent kinase 4 (CKD4) (1:5000, proteintech), anti-cyclin dependent kinase 6 (CDK6) (1:5000, proteintech), anti-cyclin D1 (1:5000, proteintech), anti-cyclin B1(1:5000, proteintech), anti-cyclin E(1:5000, proteintech) and anti-cyclin A2(1:5000, proteintech) for overnight at 4℃. Secondary antibodies were incubated for 2 h the next day. The protein bands on the membranes were detected using Enhanced Chemiluminescent (ECL, NCM Biotech).

Whole-cell lysates were obtained by re-suspending cell pellets in RIPA buffer (50 mM Tris pH 7.4, 150 mM NaCl, 1% Triton X-100) with freshly added protease inhibitor (Roche) as previously described^{47 (link),48 (link)}. Specific antibodies or pre-immune IgGs (P.I.I.) were added to and incubated with cell lysates overnight before being absorbed by Protein A/G-plus Agarose beads (Santa Cruz). Precipitated immune complex was released by boiling with 1X SDS electrophoresis sample buffer. Western blot analyses were performed with anti-BRG1 (Santa Cruz, sc-10768), anti-Cyclin B1 (Proteintech, 55004-1), anti-LTBP2 (Sigma, HPA003415), anti-KDM3A (Proteintech, 12835-1), anti-HA (Sigma, H3663), anti-FLAG (Sigma, F3165), anti-His (Invitrogen, MA1-21315), anti-GFP (Proteintech, 50430-2), anti-Myc (Santa Cruz, sc-40), and anti-β-actin (Sigma, A2228) antibodies. All experiments were repeated three times.

A western blot assay was performed using protein extracted from cultured cells and tissues. Protein samples were separated using 10% and 12% SDS-PAGE and subsequently transferred onto PVDF membranes with a pore size of 0.22μm. The membrane was blocked using 5% skim milk and a primary antibody was then incubated overnight at 4 °C on the membranes. The primary antibodies were used as follows: anti-CENPW (1:1,000, 1030382-3; Abcam, United Kingdom), anti-βactin (1:10,000; 60004-1-Ig; Proteintech, Wuhan, China), anti-Cleavedcaspase 3 (1:1,000, #9661S; Zenbio, Chengdu, China), anti-CyclinB1 (1:1,000, 60186-1-Ig; Proteintech, Wuhan, China), anti-CDK1 (1:1,000, 11554-1-AP; Proteintech, Wuhan, China), anti-Bcl-2 (1:1,000, #2872T; CST, United States), anti-P21 (1:1,000, 50599-2-Ig; Proteintech, Wuhan, China), anti-Bax (1:1,000, 50599-2-Ig; Proteintech, Wuhan, China), and anti-STAT3 (1:1,000, #9139; CST, United States), anti-p-STAT3 (Tyr705) (1:2000, #9145; CST, United States). A chemiluminescence system was employed to detect protein bands subsequent to the incubation of primary antibody probes with secondary antibodies. The scanned blots were subjected to analysis using ImageJ version 1.51 in order to ascertain the density of the bands. Protein expression was normalized by comparing it to the expression of β-actin.

Western blotting method was used in the following experiment [14 (link), 15 ], loading 25 μg of total protein lysate per lane. The following primary antibodies were used: anti-HDAC7 (1:1000, #33,418, CST), anti-c-Myc (1:1000, #5605, CST), anti-CDK1 (1:1000, 19,532-1-AP, Proteintech), anti-Cyclin B1 (1:1000, 55,004-1-AP, Proteintech), anti-CDK2 (1:1000, 10,122-1-AP, Proteintech), anti-CyclinA2 (1:1000, 18,202-1-AP, Proteintech), anti-E-cadherin (1:1000,#14,472, CST), anti-Zeb1 (1:1000, 21,544-1-AP, Proteintech), anti-α-SMA (1:1000, 14,395-1-AP, Proteintech) and anti-β-actin (1:1000, #3700, CST). The secondary antibodies used were HRP-linked anti-IgG antibodies (1:5000, Zhongshan Company, Beijing, China).