Gtx102090

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.

Immunofluorescence was performed as described previously (13 (link)). The following primary antibodies were used: anti-NFκB (GTX102090, GeneTex), anti-β-catenin (E247, Epitomics #1247-s), anti-E-cadherin (GTX100443, GeneTex), and anti-N-cadherin (TA326835, OriGene). Goat anti-rabbit secondary antibody was coupled to AlexaFluor 555 (Invitrogen). Images were obtained with DS-Ri1 Nikon camera and Eclipse80i Nikon microscope and quantifications were performed using NIS-Elements BR 4.20.00 software (Nikon).