Ab128153

The HCC-1937 cells were harvested and lysed using RIPA lysis buffer (Beyotime Institute of Biotechnology). Protein lysates were incubated with anti-KIF18B (1:1,000; ab168812, Abcam) and anti-TRIP13 antibodies (1:1,000; ab128153, Abcam) or normal rabbit IgG (1:1,000; ab172730, Abcam) and rotated overnight at 4°C for the formation of an immune-complex. The reaction mixture was then incubated with protein A/G plus-agarose beads (Santa Cruz Biotechnology, Inc.) for 2 h at 4°C. After being washed five times with cold washing buffer, the agarose beads were heated with 40 µl 10% SDS buffer to 100°C for 5 min. The samples were analyzed using western blot analysis, as described above.

Transfected or untransfected HCC-1937 cells were lysed using RIPA lysis buffer (Vazyme Biotech Co., Ltd.) according to the manufacturer's protocol. The total protein concentration was determined using a BCA Protein Quantification kit (Beyotime Institute of Biotechnology). Total protein (50 µg protein/lane) from each sample was separated via SDS-PAGE on a 12% gel and transferred onto PVDF membranes. Following blocking with 5% non-fat milk at room temperature for 2 h, all membranes were subsequently incubated with primary antibodies at 4°C overnight. Following primary antibody incubation, the membranes were incubated with HRP-conjugated anti-rabbit secondary antibody (1:10,000; ab6721, Abcam) for a further 2 h at room temperature. Protein bands were visualized using ChemiGlow detection reagents (ProteinSimple). The blots were then visualized using a FluorChem 8900 Imager and semi-quantified using ImageJ software (V1.8.0; National Institutes of Health). The following primary antibodies were purchased which were obtained from Abcam: KIF18B (1:5,000; ab168812), TRIP13 (1:5,000; ab128153), MMP12 (1:10,000; ab52897), MMP9 (1:10,000; ab76003), β-catenin (1:5,000; ab32572), c-Myc (1:1,000; ab32072), cyclin D1 (1:200; ab16663) and GAPDH (1:10,000; ab181602). GADPH was used as the loading control.