Anti human caspase 1

Total protein from kidneys and cells were extracted as previously described^{11 (link)}. Incubation was carried out with the following primary antibodies: anti-P2X7R (Santa Cruz Biotechnology, Dallas, TX, 1:200), anti-mouse NLRP3 (Adipogen, San Diego, CA, 1:1000), anti-human NLRP3 (Cell Signaling Technology, Beverly, MA, 1:500), anti-ASC (Santa Cruz Biotechnology, Dallas, TX, 1:200), anti-IL-1β (Abcam, Cambridge, MA, 1:500), anti-mouse capsase-1 (Adipogen, 1:1000), anti-human caspase-1 (Cell Signaling Technology, 1:500), anti-human cleaved caspase-1 (Cell Signaling Technology, 1:500, only for Supplementary Fig. 5c), anti-ATP5a (Proteintech, Wuhan, China, 1:1000), anti-ATP5b (Proteintech, 1:1000), anti-Tubulin (Abcam, 1:2000), and anti-GAPDH (Cell Signaling Technology, 1:2000). After incubation with the appropriate horseradish peroxidase-conjugated IgG (Cell Signaling Technology), specific signals were determined on a Tanon 5200 Chemiluminescent Imaging System (Tanon, Shanghai) by using an ECL kit (Thermo Scientific).

Nigericin, ATP, poly(dA:dT), poly(I:C), phorbol-12-myristate-13-acetate (PMA), dimethyl sulfoxide (DMSO) and ultrapure LPS were obtained from Sigma-Aldrich (Munich, Germany). Silicon dioxide (SiO₂) and Pam3CSK4 were obtained from InvivoGen (Toulouse, France). MCC950, carnosol, and geldanamycin (GA) were obtained from TargetMol (Boston, MA, USA). MitoTracker and MitoSOX were manufactured by Invitrogen (Carlsbad, CA, USA). Salmonella was a gift from Dr. Tao Li of the National Center of Biomedical Analysis. Anti-mouse Caspase-1 (1:1000, AG-20B-0042) was from Adipogen (San Diego, USA). Anti-human cleaved IL-1β (1:2000, 12242), Anti-mouse α-SMA (1:1000, 19245s), anti-human Caspase-1(1:2000, 4199S), anti-mouse IL-1β (1:1000, 12507) and anti-NLRP3 (1:2000, 15101S) were from Cell Signaling Technology (Boston, USA). Anti-ASC (1:1000, sc-22514-R) was from Santa Cruz Biotechnology (Dallas, USA). Anti-DDDK tag (1:3000, 20543–1-AP), Anti-HSP90 (1:3000, 13171-1-AP) and Anti-GAPDH (1:2000, 60004-1-1G) were from Proteintech Group (Chicago, USA).

The sample buffer was added to the cell lysates and boiled at 100°C for 10 minutes. The same amount of protein was loaded into SDS−PAGE gel wells. The gel was electrophoresed at 80 V for 0.5 hours and 120 V for 1 hour. Proteins were transferred from gels onto PVDF membranes (Millipore, Massachusetts, USA) at 90 V for 1 hour. The membranes were blocked with 5% nonfat milk at room temperature for 2 hours and then incubated overnight with the following primary antibodies in primary antibody dilution buffer at 4°C: anti-IL-1β (R&D Systems Cat# AF-401-NA, RRID: AB_416684), anti-caspase-1 (AdipoGen Cat# AG-20B-0042, RRID: AB_2490248), anti-NLRP3 (AdipoGen Cat# AG-20B-0014, RRID: AB_2490202), anti-ASC (Cell Signaling Technology Cat# 67824, RRID: AB_2799736), anti-human IL-1β (Cell Signaling Technology Cat# 83186, RRID: AB_2800010), anti-human caspase-1 (Cell Signaling Technology Cat# 3866, RRID: AB_2069051), and anti-β-actin (ZSGB-Bio Cat# TA-09, RRID: AB_2636897). The membrane was washed three times with PBST for 5 minutes each and then incubated for 1 hour at room temperature using conjugated secondary antibodies (1:5000) in blocking buffer. The membrane was washed three times with PBST for 5 minutes each time. Images were obtained after chemiluminescence visualization.

ATP, MSU, poly A:T, nigericin, and glucose were from Sigma. The human recombinant insulin was from Nova Nordisk. One Touch^® Ultra^® Blood glucose Test System was from Roche. Oridonin (s2335) was bought from Selleck. MitoSOX, ultrapure LPS, Pam3CSK4 and MitoTracker were from Invitrogen. Protein G agarose and streptavidin-coated beads were, respectively, supplied by Millipore and Pierce Biochemicals. Anti-Flag (F2555) and anti-VSV (V4888) were bought from Sigma. Anti-NLRP3 (AG-20B-0014) and anti-mouse caspase-1 (p20) (AG-20B-0042) antibodies were from Adipogen. Anti-NEK7 (SC-50756) and anti-ASC (sc-22514-R) antibodies were obtained from Santa Cruz. Anti-β-actin (P30002) was bought from Abmart. Anti-human caspase-1 was from Cell Signaling Technology. Anti-human cleaved IL-1β (A5208206) was from Sangon Biotech. Anti-mouse IL-1β (p17) (AF-401-NA) was from R&D Systems. A standard high-fat diet (D12492, 60% kcal fat) was from Research Diet Company.