Anti ccng2

Cells were harvested and lysed with radioimmunoprecipitation assay (RIPA) buffer supplemented with Protease Inhibitor Cocktail and PhosSTOP Phosphatase Inhibitor Cocktail (Roche, Basel, Switzerland). The protein concentrations of the cell lysate supernatants were quantified using a BCA Assay Kit (Beyotime, China). Equal amounts of protein samples were separated by SDS-PAGE and transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, Billerica, MA, USA). After being blocked, the membranes were incubated at 4°C overnight with one of the following primary antibodies: anti-CCNG2 (1: 1000; Abcam, USA), cyclin D1 (1: 1000; Abcam), anti-matrix metalloproteinase-2 (MMP2) (1: 1000; CST, USA), MMP3 (1: 1000; CST), anti-MMP9 (1: 1000; CST), or GAPDH (as loading control; 1: 5000; Proteintech, USA). After washing, the membranes were incubated with secondary horseradish peroxidase-conjugated anti-rabbit IgG antibody (1: 10000; Abcam) at room temperature for 1 h, with the bands subsequently visualized by chemiluminescence (DNR Bio-Imaging Systems, Jerusalem, Israel). Band intensity was quantified using ImageJ software (National Institutes of Health, USA) and was normalized to GAPDH expression.

BGC823 and AGS cells were lysed with radioimmunoprecipitation assay (RIPA) (Beyotime). Then, the lysates were separated using 10% SDS-PAGE and transferred onto appropriate nylon membranes (Sigma) and were subsequently incubated with specific antibodies overnight at 4°C (anti-CCNG2, Abcam; anti-p21, CST; anti-CDK2, ABclonal; anti-WBP11, ABclonal, or anti-glyercaldehyde-3-phostphate dehydrogenase (GAPDH), ProTech), followed by the incubation with the secondary antibody. Finally, the bands were exposed using the enhanced chemiluminescence (ECL) chromogenic substrate (Bio-Rad), and GAPDH was applied as a negative control.