Novex 4 20 tris glycine gel

Gastric tissue was homogenized in RIPA buffer (Pierce, Rockford, IL). Western blots were performed using the SDS-PAGE System. Briefly, 20 μg of protein were resuspended in sample buffer, then electrophoresed on Novex 4–20% Tris-Glycine gels (Invitrogen) with Tris running buffer; transferred to PVDF membrane using the iBlot Dry Blotting System (Invitrogen) according to the manufacturer's instructions. The membranes were incubated in 5% nonfat milk for 1 hour at room temperature and then sequentially probed with primary antibodies to GLI2 (#sc271786, Santa Cruz Biotechnology, Dallas, TX), KIF3A (Abcam, #11256), or GAPDH (Thermo Fisher, #MA5-15738) overnight at 4°C.

The excised tumors were weighed and equal size tissue was homogenized in tissue lysis buffer (TPER, Thermo Scientific, 78510) supplemented with EDTA free protease inhibitor cocktail (Thermo Scientific, 87785), with ratio of ∼0.1g of tissue to 1 mL T-PER lysis buffer. The cell debris was separated by centrifugation at 10,000g for 10 min. After discarding the pellet, the protein concentration was determined using the BCA protein assay (Pierce). 10–50 μg of cell protein samples were resolved on Novex 4–20% Tris-Glycine Gels (Invitrogen) and transferred onto polyvinylidene fluoride membranes (Invitrogen). Blots were incubated with 1:1000 rabbit polyclonal antibody against cleaved IL-1β (Cell Signaling #63124), cleaved GsdmD (Cell Signaling #50928), or β-actin (Cell Signaling #8457 for 4h at RT or for 16 h at 4°C. The membranes were washed with PBST for 5 min × 3 times and incubated with 1:15000 horseradish peroxidase-conjugated goat anti-rabbit secondary antibody (Biorad) for 2h. The signal was detected with an enhanced chemiluminescent substrate (Pierce) and membranes were imaged using iBright CL750 Imaging System (Life Technologies, A44116). The western blot bands were quantified via densitometry using the iBright Analysis Software.