Anti il 1β antibody

Murine IL-1β standard (Thermo Fisher, Scientific Waltham, MA, USA) at a concentration of 500 pg/ml and murine pro-IL-1β standard (Thermo Fisher, Scientific Waltham, MA, USA) at a concentration of 1,000 pg/ml were mixed separately with FhCL3 (1 μg/ml) or rvFhCL3 (1 μg/ml) in a reaction buffer containing 0.1 M sodium phosphate buffer, pH 6.0, 1 mM DTT and 1 mM EDTA. The samples were kept in the incubator for 2 h, at 37°C. Then, the determination of mature IL-1β was performed by capture ELISA and Western blot. For ELISA, an IL-1β Mouse Uncoated ELISA Kit (Thermo Fisher, Scientific Waltham, MA, USA) was used. For western blot anti-IL-1β antibody (Cell Signaling Technology, Danvers, MA, USA) and a mouse IL-1 beta /IL-1F2 Antibody (R&D Systems, MN, USA) were used as primary antibodies, to detect IL-1β and pro-IL-1β, respectively. IRD Fluor 800-labeled IgG (LI-COR Inc., Lincoln, NE, USA) was as and secondary antibody.

Mouse colon tissues were cut and fixed in 4% paraformaldehyde (PFA) for 24 h. Then fixed tissues were embedded in paraffin, sectioned, and stained using hematoxylin and Eosin Staining Kit (Beyotime Biotechnology). Immunohistochemistry (IHC) was performed as we described previously [35 (link)]. Briefly, tissue sections were incubated by appropriate primary antibodies overnight at 4 °C, followed by incubation with GTVisionTM III Detection System/Mo & Rb/including DAB (gene tech, Shanghai). The cell nucleus was stained with hematoxylin (Sigma-Aldrich). The stained sections were scanned with Leica versa 8. Anti-IL1β antibody was purchased from Cell Signaling Technology. Anti-c-Kit antibody was obtained from Abcam. Anti-TNFα antibody were purchased from Santa Cruz biotechnology. Anti-IL6 antibody were purchased from Servicebio.

Lipopolysaccharides (Escherichia coli O111:B4, L2630, for animal experiments; Escherichia coli O111:B4, L4391, for cell experiments) were purchased from Merck (New Jersey, USA). ACSS2 inhibitor (cat no: S8588) was purchased from Selleck (Houston, USA). ML264 (cat no: HY-19994) was purchased from MedChemExpress (New Jersey, USA). Lotus Tetragonolobus Lectin (LTL) (cat no: FL-1321-2) was purchased from Thermo Fisher (MA, USA).
The following antibodies were used. ACSS2 antibody (cat no: ab133664) from Abcam (Cambridge, UK); KIM-1 antibody (cat no: sc-518008) from Santa Cruz (California, USA); F4/80 antibody (cat no: 28463-1-AP) from Proteintech (Wuhan, China); Cleaved GSDMD (N Terminal) antibody (cat no: A22523) and NLRP3 antibody (cat no: A5652) from Abclonal (Wuhan, China); caspase-1 antibody (cat no: 24232), anti-IL-1β antibody (cat no: 12242) from Cell Signaling Technology (Massachusetts, USA); GSDMD antibody (cat no: AF4012), NF-κB p65 antibody (cat no: AF5006), phospho-NF-κB p65 (Ser536) antibody (cat no: AF2006), and KLF5 antibody (cat no: AF7542) from Affinity (Changzhou, China).

The following materials were used: anti-Bcl-2 antibody (Abcam, 182,858), anti-Bax antibody (Abcam, 32,503), anti-GAPDH antibody (Abcam, 8245), anti-cleaved caspase-3 antibody (Cell Signaling Technology, 9664), anti-IL-1β antibody (Cell Signaling Technology,12,242), anti-IL-6 antibody (Cell Signaling Technology, 12,912), anti-IL-10 antibody (Abcam, 189,392), anti-TNF-α antibody (Abcam, 183,218), anti-TLR4 antibody (Abcam, 22,048), anti-MyD88 antibody (Abcam, 133,739), anti-NF-κB antibody (Abcam, 16,502), anti-p-NF-κB antibody (Abcam, 76,302), goat anti-rabbit HRP-conjugated antibody (Abcam 6721), and goat anti-mouse HRP-conjugated antibody (Abcam 6789).

The primary antibodies used for western blotting (anti-ASC antibody, anti-Caspase-1 p20 antibody, anti-IL-18 antibody, anti-IL-1β antibody, anti-Pro-Caspase-1 antibody, anti-Pro-IL-18 antibody, anti-Pro-IL-1β antibody anti-NLRP3 antibody, anti-p-ERK antibody, anti-p-P38 antibody, anti-p-JNK antibody), in the study were all obtained from Cell Signaling Technology (1:1,000 Massachusetts, USA). The secondary antibodies used for western blotting were obtained from Beyotime (1:1,000 Jiangsu, China). Inhibitors were obtained from MCE (shanghai, China). Other chemical reagents were obtained from Dingguo Changsheng (Beijing, China).

Liver specimens or cell cultures were lysed in RIPA buffer (Thermo Fisher Scientific), supplemented with protease inhibitors (Roche) for 30 min at 4°C, and then, the samples were centrifuged at 13,000 rpm at 4°C for 10 min. The protein concentrations in the supernatants were measured with the Bradford method (Bio‐Rad, Hercules, CA, USA). Then, the proteins were mixed with SDS sample buffer and boiled for 5 min. After separation by SDS–PAGE, the proteins were transferred to nitrocellulose membranes, which were blocked with nonfat milk and incubated with primary antibodies (anti‐β‐actin antibody, anti‐caspase‐1 antibody and anti‐IL‐33 antibody: Abcam; anti‐NLRP3 antibody, anti‐P2X2 antibody, anti‐P2X4 antibody, anti‐P2X7 antibody, anti‐IL‐1β antibody, and anti‐IL‐18 antibody: Cell Signaling Technology) at 4°C overnight. The membranes were washed with TBST (20 mM Tris‐HCl, 150 mM NaCl, and 1% Tween‐20) and incubated with HRP‐conjugated secondary antibodies (1:5,000) (Abcam) for 1 h. Protein‐antibody complexes were visualized using enhanced chemiluminescence (Merck Millipore, Billerica, MA, USA), and the blots were exposed to X‐ray films. The expression of each protein was quantified as the ratio of the band intensity of each protein to the band intensity of β‐actin using Photoshop software (Adobe Systems, Incorporated, San Jose, CA, USA).