Cleaved caspase 1

Min6 cells were plated in six-well dishes with overnight culture and then incubated with 10 μmol/L tested compounds and 300 μmol/L PA for 12 h. Total proteins of cells were lysed with RIPA lysis buffer (Boster, China) and the protein lysis was separated by SDS-PAGE and transferred from gel to PVDF membrane (Millipore, USA). Then, the membranes were blocked, stained overnight with corresponding primary antibodies and then with second antibody for 1 h. The signals of detected proteins were measured by Chemidoc Imaging System (BIO-RAD, USA). The antibody used were as follows: TXNIP (Abcam, ab188865), NLRP3 (Wanlei, Wuhan, China; WL02635), Cleaved caspase 1 (Wanlei, Wuhan, China; WL03450), Cleaved caspase 3 (CST, #9664s), GAPDH (Bioworld, Nanjing, China; AP0063).

Each frozen hippocampal and cortical tissue (weighing 30-50 mg) and precooled RIPA lysis buffer (Solarbio, China), added with protein phosphatase inhibitor complex (Biomed, China) and phenylmethylsulfonyl fluoride (PMSF, BestBio, China), were mixed in a proportion of 1 : 5, ground into homogenate, and centrifuged for liquid supernatant. The supernatant was then quantified by Pierce™ BCA protein assay (23225, Thermo Fisher Scientific, Waltham, MA, USA). Protein samples were separated with 12% SDS-PAGE gel and transferred onto PVDF (Millipore, USA) membranes. The membranes were incubated with antibodies of NLRP3 (1 : 1000, Abcam, USA), pro-caspase-1 (1 : 500, Wanleibio, China), cleaved-caspase-1 (1 : 500, Wanleibio, China), ASC (1 : 1000, Abcam, USA), GSDMD (1 : 1000, Abcam, USA), CD63 (1 : 500, Wanleibio, China), CD9 (1 : 1000, Abcam, USA), and GAPDH (1 : 1000, Cell Signaling Technology, USA), respectively, overnight at 4°C. After washing by TBST, the membranes were incubated with horseradish enzyme labeling goat anti-rabbit IgG (1 : 5000, Boster, China) for 1 h at room temperature and subjected to ECL detection reagent (Millipore, USA). The protein bands were visualized by autoradiography and analyzed by ImageJ.

The cells were harvested in RIPA Lysis Buffer and lysed using ultrasound (Wanleibio, Shenyang, China). BCA Reagent was used to determine total protein content (Wanleibio, Shenyang, China). SDS-PAGE (Wanleibio, Shenyang, China) was used to separate equivalent quantities of protein extract, which was then deposited onto PVDF membranes (Millipore, USA). Cleaved-Caspase-1 (Wanleibio, Shenyang, China), cleaved-Caspase-3 (Wanleibio, Shenyang, China), cleaved-Caspase-4 (Affinity Biosciences, Suzhou, China), cleaved-Caspase-8 (Affinity Biosciences, Suzhou, China), GSDMD (Affinity Biosciences, Suzhou, China), GSDME (ABclonal, Wuhan, China), and GSDMD-N (Affinity Biosciences, Suzhou, China) were the primary antibodies employed in this test. After blocking with 5% skim milk for an hour, the membranes were incubated overnight with primary antibodies at 4°C. The membranes were then incubated with HRP-conjugated secondary antibodies (Wanleibio, Shenyang, China) and detected using an enhanced chemiluminescence substrate kit (Wanleibio, Shenyang, China) after washing.