Ecl plus western blotting system kit

Total proteins from lentivirus infected HO-8910 and OVCAR-3 cells were lysed by Lysis Buffer and the concentration was measured using BCA Protein Assay Kit (Cat. # 23225, HyClone-Pierce). After heated for 20 min at 100 °C, proteins were electrophoresed through a 10% SDS-PAGE. PVDF membranes were blocked by TBST with 5% skim milk, and then the membranes were incubated with primary antibodies and secondary antibody (detailed in Table S3) at 4 °C overnight. ECL plusTM Western blotting system kit from Amersham was used for color developing and target proteins detecting and bands were analyzed with ImageJ software.

Equal amounts of nuclear protein extracts prepared from U251 cells were incubated with the pCREB antibody, CBP antibody, or normal IgG (sc-2027X, SCBT) overnight at 4 °C. Then, the agarose-conjugated protein-A/G beads (SCBT) were added into the immunocomplex and the mixture was incubated at 4 °C for another 12 h. After extensive washing with ice-cold WB/IP lysis buffer with protease inhibitors, the beads were mixed with SDS loading buffer and boiled. The proteins in the supernatant were separated by SDS-PAGE and transferred to NC membranes. Blots were incubated with blocking reagent (TBST solution containing 5% non-fat milk), primary and secondary antibodies diluted in TBST, and then developed with enhanced chemiluminescence (ECL) + plusTM Western blotting system kit (Amersham). X-ray film was exposed to the membrane and then developed.

Total proteins lentivirus infected HCT116 and RKO cells were lysed by Lysis Buffer and the concentration was measured using BCA Protein Assay Kit (HyClone-Pierce). PVDF membranes. After blocked by TBST with 5% skim milk, the membranes were incubated with primary antibodies (detailed in Additional file 1: Table S1) at 4 °C overnight. We washed with TBST three times and added HRP-conjugated IgG polyclonal antibodies as the secondary antibody incubated for 2 h at room temperature. ECL plusTM Western blotting system kit from Amersham was used for color developing and target proteins detecting. For co-immunoprecipitation, proteins were immunoprecipitated by anti-CHSY1 or anti-MCM8 antibody, followed by western blot analysis with antibody of ubiquitin, MCM8 and NEDD4.

After infected with CCNI2 shRNA, HCT 116, and RKO cells were collected and lysed by Cell Lysis Buffer (9803S, Cell Signal Technology, Danvers, MA). BCA Protein Assay Kit (23225, HyClone‐Pierce, Logan, UT, USA) was used to measure protein concentration. The total cellular proteins (20 μg) were subjected by 10% SDS‐PAGE for western blot (WB) analysis, and transferred to polyvinylidene difluoride (PVDF, IPVH00010, Millipore Life Science, Boston, MA, USA) membranes by wet transfer. Membranes were blocked by TBST with 5% skim milk at 4°C for 1 h, and then incubated in primary antibodies (CCNI2 antibody: 1:1000, ab97767, Abcam, Cambridge, MA, USA; GAPDH antibody: 1:3000, AP0063, Bioworld, MN, USA) at 4°C overnight. After that, horseradish peroxidase (HRP)‐conjugated goat anti‐rabbit IgG polyclonal antibody (1:3000, A0208, Beyotime Biotechnology, Shanghai, China) as the secondary antibody was used to incubate the membranes for 2 h at room temperature. ECL‐Plus^TM Western blotting system kit from Amersham (RPN2232, Chicago, IL, USA) was used for color developing, and chemiluminescence imaging system (AI600, GE Healthcare Life Sciences, USA) was employed to take photos, and Image J (National Institutes of Health, Bethesda, Maryland, USA) was used for immunoblotting density analysis.

After infection, RKO and HCT–116 were collected and lysed with 1 × Lysis Buffer lysis (Cell Signal Technology, Danvers, MA). At the same time, 10% SDS–PAGE was used to segregate the total proteins and transferred into PVDF membranes followed by blocking with a blocking solution (TBST solution containing 5% skim milk) at room temperature for 1 h. Next, the membranes were incubated with antibodies and washed with TBST solution for three times (10 min/time). Finally, the ECL + plus^TM Western blotting system kit (Amersham, Chicago, IL, United States) was used for color rendering and X-ray imaging was captured.
In Co-IP analysis, HCT-116 cells were lysed, and total proteins were extracted. Then, 1.0–1.2 mg proteins were incubated with antibody overnight, followed by 2 h of incubation with 20 μL beads at 4°C. After that, the complex was lysed and incubated at 95–100°C for 10 min. Then the proteins in the immunocomplex were separated by 10% SDS-PAGE for western blot assay. Finally, the corresponding primary and secondary antibodies were incubated to identify interacting proteins. Antibodies used in western blot assay were shown in Supplementary Table 1.