Txnip

To determine protein expression levels, whole-cell or tissue extracts were lysed with RIPA peptide lysis buffer (Beyotime Biotechnology, Jiangsu, China) containing 1% protease inhibitors (Pierce) and Western blot analysis were performed according to standard procedures. Primary antibodies were used as follows: TXNIP (1:2000, Abcam), NLRP3 (1:1000, Cell Signaling Technology), ASC (1:1000, Abcam), pro-Caspase-1 (1:1000, Abcam), cleaved-Caspase-1 (1:1000, Abcam), IL-1β (1:1000, Abcam), IL-18 (1:1000, Abcam), and GAPDH (1:3000, HuaBio). Protein bands were developed using the enhanced chemiluminescence (ECL) system and were visualized using the ChemiScope Western Blot Imaging System (Clinx Science Instruments Co., Ltd). The gray value assay was performed by using Image J software (Rawak Software, Inc. Germany).

When cell confluence reached ~90%, total protein was extracted using RIPA protein lysis with PMSF (Beijing Solarbio Science & Technology Co., Ltd.). After bicinchoninic acid (BCA; Wanleibio Co., Ltd.) measurement, the proteins (40 µg/lane) were added to 10% SDS-PAGE (Nanjing KeyGen Biotech Co., Ltd.) and separated, and then transferred to PVDF membrane (EMD Millipore). After blocking with 5% non-fat milk at room temperature for 2 h, membranes were incubated overnight at 4°C with primary antibodies and secondary antibodies at room temperature for 2 h. Antibody incubations were performed at the following dilutions: Trx (cat. no. ab26320; 1:5,000; Abcam), Txnip (cat. no. 14715s; 1:1,000; Cell Signaling Technology, Inc.), ITCH (cat. no. 12117s; 1:1,000; Cell Signaling Technology, Inc.), GAPDH (cat. no. 10494-1-AP; 1:5,000; ProteinTech Group, Inc.), caspase-3 (cat. no. 19677-1-AP; 1:5,000; ProteinTech Group, Inc.) and horseradish peroxidase-conjugated secondary antibodies against rabbit (cat. no. 10285-1-AP; 1:10,000; ProteinTech Group, Inc.). The blots were visualized using ECL (Thermo Fisher Scientific, Inc.) and an ECL system (Fluor Chem FC2, Alpha Innotech, Inc.) was used to visualize the bands. Densitometric analysis was performed by ImageJ software (version 1.8.0; National Institutes of Health). GAPDH was used to normalize the expression data.

Protein samples from tissues or cells were extracted using a radioimmunoprecipitation assay buffer supplemented with proteinase inhibitors (Beyotime, Beijing, China) and separated on 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis by electrophoresis. After being transferred onto polyvinylidene fluoride membranes, the proteins were blocked with 5% skimmed milk at room temperature for 1h. Subsequently, the polyvinylidene fluoride membranes were incubated with primary antibodies against TXNIP (Abcam, Cambridge, UK) and β-actin (Abcam) at 4 °C overnight. Next, the membranes were incubated with corresponding secondary antibodies conjugated to horseradish peroxidase. The immunoreactive band were visualized using an enhanced chemiluminescence substrate kit (Pierce, Rockford, USA). β-actin was used as the internal reference.