Rapi lysate

Gastric cancer cells were lysed with RAPI lysate (Beyotime Biotechnology, Shanghai, China) and centrifuged at 4 °C for 30 min at 14,000 rpm to concentrate and collect the proteins. The concentration of proteins in each sample was determined using a BCA protein assay kit (Beyotime Biotechnology, Shanghai, China). After electrophoresis with 10% SDS-PAGE, the proteins were transferred to a PVDF membrane (Millipore, MA, US), blocked with 5% skimmed milk for 2 h, and then washed 3 times in TBST buffered saline. The PVDF membrane was cut according to protein molecular weight and experimental grouping, and they were then incubated with Bax, Bcl-2, cleaved-PARP, cleaved-caspase-3, 8, 9, cyto-c, LC3-II, Beclin1, p62, Akt, p-Akt, mTOR and p-mTOR (1:1000, Abcam, Cambridge, MA, USA) primary antibodies overnight at 4 °C. After being washed three times with TBST buffer, the membrane was incubated in IgG antibodies 1:2000 dilution (MultiSciences, Shanghai, China) at room temperature for 1 h, and then washed three times with TBST buffer salt. An ECL chemiluminescence kit (Beyotime Biotechnology, Shanghai, China) was used for color development in a dark room, and then gel images were collected and analyzed. The corresponding gray value of each image strip was analyzed using Quantity One software.

5 × 10⁶ cells at the logarithmic growth stage were collected. To extract the total proteins, RAPI lysate (Beyotime, Nanjing, China) containing protease and phosphatase inhibitors was used. The relevant protein concentration was measured using a BCA kit (Thermo Fisher Scientific, MA, USA). After quantification, the proteins were separated using SDS-PAGE. These proteins were transferred to polyvinylidene fluoride (PVDF) membranes and sealed in 5% degrease milk for 1 h. The blots were incubated at 4 ℃ overnight with the primary antibodies against ASC (CST, 67,824, 1:1000), pro-caspase-1 (Abcam, ab32499, 1:10,000), caspase-1 (Santa, sc-56036, 1:500), IL-1β (CST, 12,703, 1:1000), IL-18 (CST, 54,943, 1:1000), and GAPDH (Abcam, ab181602, 1:10,000). The membranes were washed and incubated with horseradish peroxidase (HRP)-conjugated antibodies at 23 °C for 1 h and then developed using ECL-Plus reagent (Thermo Fisher Scientific). We observed the membranes using gel imaging system and analyzed the findings.