Gtx14367

Aorta tissues were homogenized in radioimmunoprecipitation assay (RIPA; Solarbio, R0010, Beijing) lysis buffer. Equal amounts of protein (40 μg) were extracted from the total aorta and separated by 10% SDS–polyacrylamide gel electrophoresis (Solarbio, P1200, Beijing). The proteins were then transferred to polyvinylidene fluoride (PVDF; Millipore, ISEQ00010, USA) membranes and blocked with 5% skim milk powder (BD, 232100). The following primary antibodies were incubated with PVDF membranes at 4°C overnight: NF-κB antibody (1 : 2000, GeneTex, GTX102090), anti-thioredoxin interacting protein (TXNIP) antibody (EPR14774) (1 : 1000, Abcam, ab188865), NLRP3 antibody (1 : 1000, GeneTex, GTX64347), anti-ASC antibody (1 : 200, Abcam, ab175449), caspase-1 antibody (14F468) (1 : 1000, GeneTex, GTX14367), IL-1β antibody (1 : 2000, GeneTex, GTX55675), and β-actin antibody (1 : 5000, GeneTex, GTX109639). The PVDF membranes were then extensively rinsed and incubated at room temperature with secondary antibodies (1 : 5000, Proteintech, SA00001-1, SA00001-2, Wuhan) for 1 h. A biomolecular imager (Fuji, LAS-4000, MINI, Japan) and QualityOne were used to scan and quantitate the membranes. All experiments were repeated 3 times.

After protein was extracted from renal tissues and cells, protein concentration was determined using the BCA method. After denaturation, 30–80 μg protein was separated using 12% SDS-PAGE. Proteins were transferred to PVDF membranes using wet transfer. The membranes were blocked in 5% skim milk at room temperature for 1.5 h, and then incubated overnight at 4°C with the following primary antibodies specific for: IL-1β (1:1000, #12242, Cell Signaling, Shanghai, China); IL-18 (1:1000, ab71495, Abcam, Shanghai, China); caspase-1 (1:500, GTX14367, GeneTex, United States); GSDMD (1:500, orb390052, Biorbyt, Cambridge, United Kingdom); and β-actin (1:5000, AC004, ABclonal, Wuhai, China). The membranes were then washed and incubated at room temperature in the dark for 2 h with the following secondary antibodies: anti-mouse IgG (H + L, DyLight^TM 800 Conjugate, 1:15000, #5257, Cell Signaling, Shanghai, China) and anti-rabbit IgG (H + L, DyLight^TM 680 Conjugate, 1:15000, #5366, Cell Signaling, Shanghai, China). A two-color infrared fluorescence imaging system was used to visualize protein bands. Protein concentration in the bands was quantified using ImageJ and normalized against that of β-actin, used as loading control.