Nlrp3

Western blotting analysis was performed as previously described (Wang et al. 2017 (link)). Briefly, total proteins from cultured cells were obtained, separated by SDS–PAGE electrophoresis and transferred onto nitrocellulose membranes. Primary antibodies against IL-1β (1:1000, CST), NLRP3 (1:500, Proteintech), NF-κB (1:1000, CST), p-NF-κB (1:1000, CST), Atp5a1 (1:2000, Proteintech), Ndufv2 (1:5000, Proteintech), β-actin (1:10,000, Proteintech) and appropriate secondary antibodies were used. The optical density of bands was analyzed by ImageJ software. The expression levels of the examined proteins were normalized to those of β-actin.

The extraction of total myocardial tissue proteins was completed using RIPA lysis buffer (Solarbio, China), followed by the determination of protein concentration using a BCA kit (Solarbio, China). The proteins were denatured at high temperature, separated by SDS-PAGE electrophoresis, and then transferred to PVDF membranes (Millipore, USA). After completing the closure (60 min) at room temperature using non-fat milk, the proteins were incubated overnight at 4 ℃ with SIRT1 (dilution rate 1:1000, Abcam, USA), AMPK (dilution rate 1:1000, Abcam, USA), p-AMPK (dilution rate 1:1000, Abcam, USA), NLRP3 (dilution rate 1:1000, proteintech, China), GSDMD-N (dilution rate 1:1000, Cell Signaling Technology, USA), ASC (dilution rate 1:1000, Abcam, USA), IL-18 (dilution rate 1:1000, Abcam, USA), caspase-1 p20 (dilution rate 1:1000, Bioss, China), IL-1β (dilution rate 1:1000, Abcam, USA), and GAPDH (dilution rate 1:10000, Abcam, USA) primary antibodies. The proteins were incubated with secondary antibodies (dilution rate 1:10000, Santa Cruz Biotechnology, USA) at room temperature for 1 h the next day, and the protein bands were developed under the ultrasensitive automatic chemiluminescence imaging analysis system (FluorchemM, Protein Simple, USA). The detection of relative protein expression was completed using Image J (NIH, MD, USA) software.

Protein samples were separated by 8%, 10%, or 12% sodium dodecyl sulfate-polyacrylamide gels (SDS-PAGE) and then transferred onto PVDF membranes (EMD Millipore, Burlington, MA, USA). After being blocked with 5% non-fat milk, the membranes were incubated with the following primary antibodies at 4°C overnight: α-tubulin (GeneTex, Irvine, CA, USA), collagen I (GeneTex), α-SMA (Proteintech, Chicago, IL, USA), TGF-β (Cell signaling technology, Danvers, MA, USA), TNF-α (GeneTex), NLRP3 (Proteintech), caspase-1 (Proteintech), IL-18 (Proteintech), IL-1β (Proteintech), caspase-3 (GeneTex), caspase-8 (GeneTex), caspase-9 (GeneTex), and BCL-2 (GeneTex). Membranes were then incubated with HRP-conjugated mouse anti-IgG (EMD Millipore) or HRP-conjugated rabbit anti-IgG (EMD Millipore) secondary antibodies for 1 h. Membranes were developed using ECL detection reagent (EMD Millipore). Relative protein levels were quantified using Image J (Version 1.46, National Institute of Health, Bethesda, MD, USA), and protein densitometry were expressed relative to that of α-tubulin.

The total protein of the PC12 (n = 5 for each group) or hippocampus (n = 3 for each group) was extracted and separated on a 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) with a constant voltage of 75 V. The electrophoresed proteins were transferred to nitrocellulose membranes with a transfer apparatus (Bio-Rad, Hercules, CA, USA). The membranes were blocked with 5% nonfat milk in Tris-buffered saline (pH 7.5) with 0.05% Tween 20 for 2 hours at room temperature. Primary antibodies, including HSF1, NLRP3, IL-1, caspase1, and GAPDH (Proteintech, Chicago, IL, USA), were diluted to 1 : 1000 in TBS buffer overnight at 4°C. The blots were washed 3 times in TBS buffer for 10 min and then immersed in the secondary antibody solution containing the goat-IgG rabbit polyclonal antibody (1 : 5000 in TBS buffer, Proteintech) for 2 h and diluted with TBS buffer. The membranes were washed 3 times for 10 min in TBS buffer. The immunoblotted proteins were visualized using an ECL Western blot luminol reagent (Advansta, Menlo Park, CA, USA) and quantified using a Universal Hood II chemiluminescence detection system (Bio-Rad) by imaging scans.

Tissue sections from mouse skin were deparaffinized, hydrated, antigen retrieval, serum blocking, incubation of primary antibody (NLRP3; Proteintech) and corresponding secondary antibodies, washing, diaminobenzidine color development, hematoxylin counterstaining, and mounting of the sections were performed sequentially. The expression and localization of NLRP3 in skin tissues were detected in three randomly selected high‐power fields per section under a microscope.

Total protein was extracted from cells or tissues with the use of RIPA Vs protease inhibitor cocktail (100:1), while cytoplasmic proteins and nucleoproteins were extracted by employing extraction kits (BOSTER, China). Protein concentration was quantified based on BCA kit (Beyotime, China). Equal proteins (15–25 μg) were separated on 10%–12% Sodium dodecyl sulfate (SDS) gels and then transferred onto Polyvinylidene fluoride (PVDF) (0.22/0.45 μm) membranes. After being blocked with 5% skim milk, the membranes were incubated with primary antibodies (1:1,000) overnight at 4°C. Primary antibodies consisted of Sucnr1 (Abcam, ab272856), Hif-1α (CST, #36169), IL-1β (CST, #12242), NLRP3 (Proteintech, #19771-1-ap), NF-κB (Proteintech, 66535-1-lg), Lamin B1 (Abcam, ab16048), Caspase-1 (Proteintech, 22915-1-ap), and β-Actin (Affinity, AF7018). Subsequently, the membranes were incubated with secondary antibody [goat anti-rabbit IgG HRP/goat anti-mouse IgG Horseradish Peroxidase (HRP) (1:10,000, ZSGP-BIO, China)] at Room temperature (RT) for 1 h. Bands were visualized with Enhanced chemiluminescence (ECL) reagent (MilliporeSigma, USA) and then captured on FluoChem E or ChemiDoc Imaging System. The optical density of bands was analyzed with the use of ImageJ software.

Total protein was extracted from endothelial cells using the same procedures as described in detail elsewhere^{41 (link)}. The protein concentrations were determined by BCA Protein Assay kit (Bio-Rad, Mississauga, ON, Canada). Equal amounts of protein lysates were separated by SDS-PAGE and transferred onto nitrocellulose membranes followed by block with 5% skimmed milk at room temperature for 2 h. Subsequently, the membranes were incubated with the primary antibodies against NLRP3 (Proteintech, Chicago, USA, 1:1000, Cat. No.: 19771-1-AP), ASC (Santa Cruz, USA, 1:500, Cat. No.: sc-22514-R), Caspase-1 (Proteintech, Chicago, USA, 1:1000, Cat. No.: 22915-1-AP), IL-1β (ABclonal, Boston, USA, 1:1000, Cat. No.: A1112), IL-18 (ABclonal, Boston, USA, 1:1000, Cat. No.: A1115), or GAPDH (Proteintech, Chicago, USA, 1:2000, Cat. No: 60004-1-lg) at 4 °C overnight. After washing with PBST three times, the membranes were incubated with the fluorescence-conjugated anti-rabbit IgG secondary antibody (1:10,000) for 1 h. Western blot bands were examined and analyzed by Odyssey Imaging System (LI-COR, Inc., Lincoln, NE, USA).