Sc 81868

Proteins were extracted from the ileum as previously described [24 (link)]. The primary antibodies were as follows: Anti-claudin 1 (1:500 dilution, 13050-1-AP, Proteintech Group, Chicago, IL, USA), anti-occludin (1:1000 dilution, ab31721, Abcam, Cambridge, UK), anti-caspase-1 (1:200 dilution, sc-56036, Santa Cruz Biotechnology, Dallas, TX, USA), anti-caspase-4 (1:1000 dilution, GTX113639, GeneTex, San Antonio, TX, USA), anti-GSDMD (1:500 dilution, sc-81868, Santa Cruz Biotechnology) and anti-Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), (1:1000 dilution, 60004-1-Ig, Proteintech Group). Horseradish peroxidase-conjugated secondary antibodies used were goat anti-mouse IgG (1:5000 dilution, SA00001-1, Proteintech Group) or goat anti-rabbit IgG (1:5000 dilution, SA00001-2, Proteintech Group). The bands were visualized using a Tanon-5200 gel image system (Tanon, Shanghai, China). The intensity of bands was quantified by densitometric analysis using ImageJ software (National Institutes of Health, Bethesda, MD, USA).

The total protein was extracted from placental tissues and HTR-8/SVneo cells using RIPA lysate (Beyotime, China) (with PMSF-protease and phosphatase inhibitor added), and then the protein concentration was measured by the BCA assay kit (Beyotime, China) according to the instructions of manufacturer. The protein samples of 30ug per well were added to 10% of the twelve-alkyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) slot to separate the protein and then electrotransferred onto the PVDF membranes. Next, the nonspecific site of the membranes were blocked with 5% skimmed milk for 1 h at room temperature and incubated overnight at 4 ℃ with first antibodies NLRP3 (1:1000, ab214185, Abcam), Caspase-1 (1:500, ab62698, Abcam), GSDMD (1:500, sc-81,868, Santa Cruz) and β-actin (1:1000, AF0003, Beyotime). On the second day, the membranes were incubated with the horseradish peroxidase (HRP) labeled secondary antibodies (1:1000, A0208/A0216, Beyotime) at room temperature for 1 h. Finally, under the action of ECL luminescent liquid, immunoreactive signals were detected by the Bio-Rad gel imaging system (MG8600 Thmorgan Biotechnology Co., Ltd., Beijing, China). The IPP6.0 software (Media Cybernetics, Singapore) was used to analyze the gray values and calculate the relative protein levels of NLRP3, Caspase-1 and GSDMD. The experiment was repeated at least three times.

Lysis buffer containing 10 mM Tris-buffer (pH 7.6), 1% Triton X-100, 1% phosphatase inhibitor cocktail, and 1 mM PMSF was used to lyse cells. Cell lysates were then boiled in SDS sample buffer and resolved on a 10% SDS-PAGE gel. Immunoblots were incubated overnight with primary antibodies against NLRP3 (AG-20B-0014-C100, Adipogen, CH), ASC (AG-25B-0006, Adipogen, CH), mouse caspase-1 (AG-20B-0042-C100, Adipogen, CH), human caspase-1 (AG-20B-0048-C100, Adipogen, CH), IkBa (4814, Cell Signaling, USA), phospho-IkBa (2859, Ser32) (Cell Signaling, USA), A20 (23456-1-AP, Proteintech, USA), IL-1β (12242, Cell Signaling, USA) human GSDMD (sc-81868, Santa Cruz Biotechnology, USA), mouse GSDMD (ab209845, Abcam, USA), and GAPDH (10494-1-AP, Proteintech, USA). Immunoblots were examined using an ECL detection reagent (Millipore Corporation, Billerica, MA, USA).