Mouse anti human hax 1

Cells were collected and homogenized in 1× cell lysis buffer (Solarbio) supplemented with 1 mM phenylmethylsulfonyl fluoride, phosphatase inhibitor, and complete protease inhibitor mixture (Solarbio) for 30 min on ice. They were then centrifuged at 11,000 rpm for 10 min at 4°C. The supernatant was assessed using a Bicinchoninic Acid Protein Assay Kit (Solarbio). After measurement, the samples were combined with 5× sodium dodecyl sulfate loading buffer and boiled at 100°C for 10 min. For each protein, equal amounts of samples (20–100 μg) from each group were analyzed by SDS–polyacrylamide gel electrophoresis as previously described. After proteins were transferred onto a polyvinylidene difluoride membrane, the membrane was incubated with 5% BSA at room temperature for 2 h to block the non-specific protein site, and then with corresponding primary antibodies (mouse-anti-human HAX-1 from BD, Hsp90 from Proteintech; Rabbit anti human p21, p53, Bax, ubiquitin from Proteintech, Caspase-3, Caspase-9, PARP, MDM2, pMDM2, Akt1, pAkt1 from CST, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and β-actin from ZSGF-BIO, China) at 4°C overnight. This step was followed by incubation with HRP-conjugated secondary antibodies (ZSGF-BIO). Visualization was achieved using a SuperSignal West Pico Trial Kit (Thermo).

Cells were collected and lysed in cell lysis buffer (Solarbio) for 30 min on ice and then centrifuged at 12,000 rpm (15 min, 4 °C). The supernatant was collected and the concentration of total protein was determined with a bicinchoninic acid protein assay (BCA)Kit (Solarbio). The samples were mixed with 5× SDS loading buffer (Solarbio) and boiled at 100 °C for 10 min. An equal amount of each sample was then separated by SDS–PAGE and the proteins were transferred onto a polyvinylidene difluoride membrane. The membranes were then incubated with 5% BSA at room temperature for 1–2 h, followed by primary antibodies (mouse-anti-human HAX-1 from BD; from Proteintech; Rabbit anti-mouse NLRP3, ASC, Caspase1, Cleaved Caspase1, GasderminD, Cleaved GasderminD, IKKε, pIKKε, TBK1 and pTBK1 from CST; β-actin from Bioworld Technology) incubation at 4 °C for 12 h. Membranes were then washed three times with TBST and incubated with HRP-conjugated secondary antibodies (ZSGF-BIO). Visualization was achieved using a SuperSignal West Pico Trial Kit (Thermo). Relative intensities of the bands were analyzed by Image-J software. Three individual biological replicates were performed in a Western blotting assay.