Anti nlrp3

Paraffin-embedded kidney sections were used to assess colocalization of caspase-1 and GSDMD or NLRP3. First, sections were subjected to microwave-based antigen retrieval. Then, tyramide signal amplification (TSA) was performed as previously described (Faget and Hnasko 2015 (link)). Briefly, the following steps were performed: 1) incubating sections with anti-NLRP3 (1:1,000, Proteintech) or anti-GSDMD (1:500, Proteintech) at 4°C overnight, 2) incubating with horseradish peroxidase (HRP)-conjugated secondary antibody for 50 min at room temperature, 3) reacting with CY3-TSA (Servicebio, China) for 10 min in the dark, 4) removing nonspecific binding antibodies by microwave treatment, 5) incubating with anti-caspase-1 (1:50, Proteintech) at 4°C overnight, 6) incubating with Alexa Fluor 488-conjugated goat anti-rabbit IgG (Servicebio, China) for 50 min in the dark, and 7) staining with DAPI solution for 10 min. Images were captured by fluorescence microscopy using excitation wavelengths of 510–560 nm (red) and 465–495 nm (green).

Proteins from mammary tissue samples were extracted for Western blotting assay. The following primary antibodies included rabbit polyclonal anti-NLRP3 (1:2000 dilution, 19771-1-AP), rabbit polyclonal anti-ASC (1:500 dilution, 10500-1-AP), rabbit polyclonal anti-ATG5 (1:1000 dilution, 10181-1-AP), rabbit polyclonal anti-ATG16L1 (1:1000 dilution, 19812-1-AP), rabbit polyclonal anti-sequestosome 1 (SQSTM1) (1:500 dilution, 18420-1-AP) (ProteinTech Group, Rosemont, IL, USA), rabbit polyclonal anti-Claudin-3 (1:500 dilution, abs130066) (Absin Bioscience, Shanghai, CHN), rabbit polyclonal anti-LC3A/B (1:1000 dilution, 12741) (Cell Signaling Technology, Danvers, MA, USA), rabbit polyclonal anti-Caspase-1 (1:1000 dilution, ab179515), rabbit polyclonal anti-ZO-1 (1:50 dilution, ab59720), and rabbit polyclonal anti-Occludin (1:8000 dilution, ab216327) (Abcam, Cambridge, UK). To verify equal sample loading, the membrane was incubated with mouse anti-β-actin (1:5000 dilution, 66009-1-Ig), mouse anti-GAPDH (1:5000 dilution, 60004-1-Ig) and rabbit anti-β-tubulin (1:1000 dilution, 10094-1-AP). HRP-conjugated anti-mouse IgG (1:5000 dilution, SA00001-1) or anti-rabbit IgG (1:5000 dilution, SA00001-2) (ProteinTech Group, Rosemont, IL, USA) were used as secondary antibodies.

After 24 h reperfusion, the rats were deeply anesthetized, and their brains were quickly removed following cardiac perfusion with 200 ml normal saline. The levels of pyroptosis-related proteins and Aβ_1-42 oligomers were detected by Western blotting. In brief, after concentrations measurement and protein denaturation, equal amounts of protein samples extracted from ischemic penumbra and equivalent area under sham were subjected to 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred onto the polyvinylidnene fluoride membranes (Millipore, Billerica, MA, USA). Subsequently, the membranes were blocked at room temperature with 5% bovine serum albumin (BSA) for 1 h and incubated with the following primary antibodies at 4°C overnight: anti-GSDMD, anti-β-actin (CST, Danvers, MA, USA), anticaspase-11, anti-IL-6, anti-IL-1β (Santa Cruz, Dallas, TX, USA), anti-NLRP3, anticaspase-1 (Proteintech, Rosemont, IL, USA), and anti-Aβ_1–42 (Abcam, Cambridge, UK). Then, the membranes were washed and incubated with corresponding secondary antibody (SAB, College Park, MD, USA) for 1 h at room temperature. After developing by the enhanced chemiluminescence kit (Millipore), pictures were captured with a gel imaging instrument (BioRad Laboratories, USA), and the intensities were analyzed by ImageJ software (National Institutes of Health, USA).

Protein samples were extracted from liver tissues and cultured cells as previously reported [28 (link)]. Equal amounts of protein (30–40 μg) were separated by 8%–15% SDS-PAGE and then transferred onto PVDF membranes. For immunoblot analysis of IL-1β levels in the cell culture medium, supernatants were collected for protein extraction as previously reported [35 (link)]. The following primary antibodies were used: anti-IL-1β (number 12242), anti-AMPK (number 2532), anti-phospho-AMPK Thr¹⁷² (number 2535), anti-phospho-GSK3β Ser⁹ (number 5558), anti-acetyl coenzyme A carboxylase (ACC, number 3676), anti-ACC Ser⁷⁹ (number 3661) (Cell Signaling Technology, Beverly, MA, USA), anti-phospho-Akt Ser⁴⁷³ (number 2118-1) (Epitomics, Burlingame, CA, USA), anti-caspase-1 (number 22915-1-AP), anti-NLRP3 (number 19771-1-AP), anti-GSK3β (number 22104-1-AP) (Proteintech Group, Chicago, IL, USA), anti-TXNIP (number sc-67134) (Santa Cruz Biotechnology, Santa Cruz, CA, USA), and anti-Akt (number A0001)(ABclonal Biotech Co. Ltd., Cambridge, MA, USA). Goat anti-rabbit (number A21020) and goat anti-mouse (number A21010) secondary antibodies were purchased from Abbkine (Redlands, CA, USA).

Western blotting was performed as previously described [56 (link)]. Cultured cells and dissected tissues were collected, and protein was extracted using RIPA lysis buffer (Beyotime, Shanghai, China) containing a cocktail protease inhibitor (Biotool, Houston, TX, USA). Samples were incubated at 99 °C for 5 min and separated by electrophoresis on a 10% SDS-PAGE gel at 115 V for 1.2 h. Proteins were transferred to a PVDF membrane at 200 mA for 1 h. Membranes were blocked with 5% milk in TBST at RT for 1 h and incubated overnight at 4 °C with the following primary antibodies: anti-NLRP3, anti-ASC, anti-caspase1, anti-CXCR2 (ProteinTech, Chicago, IL, USA), anti-GAPDH, anti-α-SMA (Santa Cruz Biotechnology, Santa Cruz, CA, USA). HRP-conjugated goat anti-mouse IgG and goat anti-rabbit IgG were used as secondary antibodies. Blots were scanned using a Clinx Science Instrument. All specific bands were quantified with an Automated Digitizing System (ImageJ 1.8.0).

HRECs obtained from passage 6 were grown to 70–80% confluence and then starved for 12 h in 0.5% FBS/ECM. HRECs were pretreated with Mcc950 for 2 h before stimulation with high glucose. Then, HRECs and proliferative membrane samples from DR patients or donor eyes were lysed using a nuclear and cytoplasmic protein extraction kit (Beyotime, Haimen, China). Lysates were centrifuged at 15 000 × g for 10 min at 4 °C. Protein was quantified using Bradford’s reagent with bovine serum albumin as a standard. Proteins were separated using 10% SDS-PAGE and transferred to a polyvinylidene difluoride membrane (Millipore, IPVH00010, Bedford, MA, USA). Then, the membrane was blocked for 1 h at room temperature with 5% (v/v) nonfat dry milk. After three washes with PBST, the membrane was incubated in PBS at 4 °C (overnight) with anti-caspase 1 (1:1000, Cell Signaling Technology, Boston, MA, USA, No.2225), anti-NLRP3 (1:1000, Proteintech, Chicago, USA, No. 19771-1-AP), anti-IL-1β (1:1,000, Cell Signaling Technology, No. 12242). The membrane was again washed with PBST and incubated for 1 h at room temperature with a horseradish peroxidase-conjugated secondary antibodies. Signals were developed using a standard ECL western blot detection reagent (Amersham Biosciences, Arlington Heights, IL, USA). Densitometric analysis was performed with ImageJ software.

Paraffin-embedded HNSCC tumor tissue sections (4 μm) were treated with xylene for deparaffinization, rehydrated with an ethanol gradient, and treated for 20 min with 3% hydrogen peroxide. Following antigen retrieval, samples were blocked with normal goat serum and then probed overnight with anti- NLRP3 (Proteintech, 19771-1-AP). An HRP-polymer anti-rabbit Kit and a DAB Detection Kit (Fuzhou Maixin Biotech, Fuzhou, China) were then used to stain samples, and hematoxylin was employed for counterstaining. An ethanol gradient was then used to dehydrate samples, which were clarified with xylene and mounted using neutral gum.