Anti asc

Treated cells were lysed in RIPA containing protease inhibitor (PMSF, 1 : 100) on ice for 30 minutes. The cell lysate was then centrifuged at 12000 rpm at 4°C for 15 min. The supernatant was then transferred to new centrifuge tubes. The protein concentrations were determined using a BCA kit (Beyotime Biotech, Shanghai, China); equal amounts (40 μg) of protein samples were separated using SDS-PAGE and then transferred to polyvinylidene fluoride (PVDF) membranes. The membranes were blocked with 5% skimmed milk powder in TBST for 2 h. The membranes were probed overnight at 4°C with anti-NLPR3 (1 : 1000, Abcam, UK), anti-ASC (1 : 1000, Proteintech, IL, USA), anti-Caspase1 (1 : 1000, Proteintech, IL, USA), and anti-IL-1β antibodies (1 : 1000, Proteintech, IL, USA). Further, the membranes were incubated with HRP-conjugated secondary antibodies(1 : 2000, Proteintech, IL, USA) for 2 h at room temperature. The antibody binding was detected using the enhanced chemiluminescence (ECL) detection kit (Beyotime Biotech, Shanghai, China).

MAC-T cells were lysed in RIPA buffer containing a protease/phosphatase inhibitor cocktail on ice for 30 min.). Protein (equal amounts, 20 µg) were loaded on 10% or 12% SDS-polyacrylamide gels and transferred to polyvinylidene difluoride (PVDF) membranes (Roche). After blocking with 5% skim milk at 37°C for 1 h and the membranes were incubated with the following primary antibodies at 4°C overnight: anti-LC3 I/II (1:1000, #4108) and anti-cleaved-Caspase-3 (1:1000, #9664) from Cell Signaling Technology (Danvers, USA); anti-ATG5 (1:750, 10181-2-AP), anti-Beclin-1 (1:1000, 11036-1-AP), anti-p62/SQSTM1 (1:1000, 18420-1-AP), anti-ASC (1:1000, 10500-1-AP), anti-BAX (1:1000, 50599-2-Ig), anti-Bcl-2 (1:1000, 12789-1-AP), anti-Caspase-3 (1:1000), anti-PINK1 (1:1000, 23274-1-AP), anti-Parkin (1:1000, 14060-1-AP), anti-GAPDH (1:5000, 60004-1-AP) and anti-β-Actin (1:5000, 60008-1-AP) from Proteintech Group Inc (Rosemont, IL 60018, USA); anti-NLRP3 (1:500, AF2155) from Beyotime Biotechnology (Shanghai, China) and anti-Caspase-1 (1:1000, ab179515) from Abcam (Cambridge, UK). The immunoreactive bands were visualized with an ECL detection system (Tanon 6200 chemiluminescence imaging workstation, Tanon Science & Technology Co., Ltd. Shanghai, China). The protein bands were quantified by densitometry using ImageJ software (version 1.50).

MCC950, a small molecule NLPR3 inhibitor, and olaparib were purchased from Selleck Chemicals (Houston, TX); oxLDL was purchased from Anhui Yiyuan Bio-technology (Bozhou, China); HyClone RPMI 1640 and DMEM were purchased from Cytiva (Shanghai, China); fetal bovine serum (FBS) and MitoSox Red were purchased from Thermo Fisher Scientific (Beijing, China); aprotinin and leupeptin were purchased from Solarbio Life Science (Beijing, China); DTT (dithiothreitol) was purchased from Wako Pure Chemical Industries (Osaka, Japan); protein A/G-agarose was purchased from Santa Cruz Biotechnology (Shanghai, China); anti-vascular cell adhesion molecule (VCAM)-1, anti-caspase-1, anti-RelA (phosphor-S536), and anti-vinculin antibodies were purchased from Abcam; anti-IL-1β and anti-IL-18 antibodies were purchased from Sab Biotech (Nanjing, China); anti-NLRP3, anti-RelA and anti-phosphor-IκВα (ser 32) antibodies, and a NF-κВ non-canonical pathway antibody sampler kit including anti-IKKα, anti-RelB and anti-p52 antibodies were purchased from Cell Signaling Technology (Shanghai, China); anti-ASC, anti-IκВα, and anti-p50 antibodies were purchased from Proteintech Group (Wuhan, China). Other reagents, unless otherwise stated, were purchased from Sigma-Aldrich (Shanghai, China).

The total protein was extracted from colon tissues using RIPA lysis buffer and quantified using a bicinchoninic acid (BCA) protein assay kit (Thermo Fisher Scientific, MA, USA). Equal amounts of protein were loaded on sodium dodecyl sulfate-polyacrylamide gel electrophoresis gel and then blotted to polyvinylidene difluoride membrane. Membranes were blocked with 5% skimmed milk for 2h and overnight incubated with primary antibodies at 4°C, followed by incubation with HRP-conjugated secondary antibody (Abcam, #ab6721, #ab6789) for 2h at room temperature. Protein signals were detected with ECL kit (Bio-Rad, CA, USA) and visualized using Bio-Rad Chemi XRS imaging system (Bio-Rad). The primary antibodies used were purchased from the following suppliers: anti-β-actin (Proteintech, #20536-1-AP), anti-ZO-1 (Thermo Fisher Scientific Inc., #61-7300), anti-Occludin (Thermo Fisher Scientific Inc., #40-4700), anti-Claudin1 (Thermo Fisher Scientific Inc., #51-9000), anti-NLRP3 (Proteintech, #19771-1-AP), anti-ASC (Proteintech, #10500-1-AP), anti-Caspase1 (Santa Cruz, #14F468), anti-ATG5 (Proteintech, #10182-2-AP), anti-ATG7 (Proteintech, #10088-2-AP), anti-p62 (Cell Signaling Technology, #5114), and anti-LC3 I/II (Affinity Biosciences, #AF5402). Band density of target protein was quantified after normalization to β-actin using ImageJ v1.8.0 software.

Western blot analysis was performed according to standard procedures, as previously described [21 (link)]. The following primary antibodies were used: anti-NLRP3 (1:1000, Abcam), anti-ASC (1:1000; Proteintech), anti-GSDMD (1:1000; Abcam), anti-CASP1 (1:1000, Proteintech), anti-DPYSL4 (1:1000; Abcam), anti-p53 (1:1000; Santa Cruz), and anti-β-actin (1:5000; Proteintech). Following incubation with the primary antibody, the membranes were washed and probed with secondary antibodies (1:5000; Proteintech) for 1 h. Proteins were visualized by chemiluminescence.

Paraffin-embedded sections were employed to deparaffinize and rehydrate the tissues. Then sections were microwaved in 0.01 M citrate buffer and washed with PBS for 15 min, and then treated with blocking buffer containing 5% BSA for 20 min at room temperature. Afterwards, the sections were incubated for 1 h at 37 °C with rabbit polyclonal antibodies anti-NLRP3 (1:1000) (Proteintech, Chicago, IL,USA), anti-ASC (1:500) (Proteintech, Chicago, IL,USA), and anti-caspase-3 (1:500) (Abcam, Cambridge, MA, US). After washing with PBS for 3 times, the secondary antibody was added, and immunostaining was performed using a DAB kit (Invitrogen, GIBCO, Carlsbad, CA, USA), and counterstain hematoxylin (Amresco, Solon, HO, USA). The ASC positive area and NLRP3 positive area were measured using ImageJ (U.S. National Institute of Health, Bethesda, MD).