Ab230302

The aforementioned paraffin samples were sectioned and immersed in 3% methanol H₂O₂. Next, the sections were retrieved in antigen retrieval solution, followed by sealing in normal goat serum blocking solution at room temperature for 20 min. Subsequently, the sections were probed with the primary anti-rabbit polyclonal anti-GLS antibody (ab260047, dilution ratio of 1: 200, Abcam, Cambridge, UK), GDH (ab170895, dilution ratio of 1: 100, Abcam), RUNX3 (ab224642, dilution ratio of 1: 1000, Abcam) and FBXO4 (ab230302, dilution ratio of 1:1000, Abcam) at 4 °C overnight. The following day, the sections were incubated with the secondary antibody IgG (goat anti-rabbit, ab6721, dilution ratio of 1: 1000, Abcam) at 37 °C for 20 min. Afterwards, the sections were stained with DAB (ST033, Weijia Technology) development, and then counter-stained with hematoxylin. Thereafter, the sections were visualized under a microscope (CX43, Olympus Optical Co., Ltd., Tokyo, Japan). The Nikon image analysis software was utilized to document the positive cells. A total of 5 non-repetitive visual fields of equal area (200 times) were selected from each section and the number of positive cells and their proportions were calculated, with the average value calculated.

RIPA lysis buffer (Beyotime) was adopted for total protein content extraction, with protein concentration assessed using BCA kits. Next, the proteins were separated by SDS-PAGE, and then transferred onto PVDF membrane (IPVH85R, Millipore, Darmstadt, Germany). After blocking, the membranes were incubated with specific primary antibodies, rabbit anti-human GLS (ab260047, dilution ratio of 1: 250, Abcam), GDH (ab170895, dilution ratio of 1: 100, Abcam), FBXO4 (ab230302, dilution ratio of 1:1000, Abcam), and GAPDH (ab181602, dilution ratio of 1:10,000, Abcam) at 4 °C overnight. The following day, the membranes were incubated with HRP-labeled IgG (goat anti-rabbit, ab205718, dilution ratio of 1: 5000, Abcam) for 1 h at room temperature. ECL was adopted to visualize the results, with band intensities quantified using the ImageJ software (1.48 u, National Institutes of Health, Bethesda, Maryland). Relative protein expression = gray values of the target protein band / gray values of internal reference GAPDH band.