Ab2105

IL-1α antibody (SC-7929) was purchased from Santa Cruz Biotechnology. Antibodies directed against phospho-p38 MAPK (4511), p38 MAPK (8690), phospho-JNK (9251), JNK (9252), phospho-ERK (4370), ERK (4695), phosphor-AKT (9611) and AKT (4691) were purchased from Cell Signaling Technology. The antibody for human S100A7 (11141-RP02), soluble recombinant human S100A7 (11141-HNAE), IL17A (12047-HNAE), IL-22 (13059-HNAE), IL-33 (10368-HNAE), IL36γ (10124-HNAE) and TNFα (10602-HNAE) were purchased from Sino Biological Inc. The antibody against β-actin (ab8227), IL-1β (ab2105), RAGE (ab54741) and calpain-1 (ab28258) were purchased from Abcam. Antibody against mS100a7a15 (AP52284) was purchased from Abgent. The inhibitors including SB202190 (S7067), SP600125 (S5567), LY294002 (L9908) and caspase-8 inhibitor (C1230) were purchased from Sigma-Aldrich. PD151746 (S7424) was purchased from Selleck.

Total proteins were lysed in RIPA lysis buffer (Beyotime Biotechnology, Shanghai, China), and the concentration of the protein samples was measured using a bicinchoninic acid (BCA) protein assay kit (Beyotime). Proteins were subjected to SDS-PAGE and then electrophoretically transferred onto PVDF membranes. Membranes were incubated with primary antibodies overnight at 4°C after blocking in 5% nonfat milk in TBST buffer, which was then coincubated with horseradish peroxidase (HRP)-conjugated anti-mouse/anti-rabbit IgG (Thermo Fisher Scientific). Proteins were visualized using a detection system of enhanced chemiluminescence (ECL), and the bands were analyzed using BandScan ImageJ software. The primary antibodies used in this study were as follows: anti-CHOP (diluted 1 : 1000, ab11419, Abcam), anti-caspase 12 (diluted 1 : 1,000, ab62484, Abcam), anti-GRP78 (diluted 1 : 100, ab21685, Abcam), anti-NLRP3 (diluted 1 : 500, ab214185, Abcam), anti-ASC (diluted 1 : 1000, ab155970, Abcam), anti-pro-IL-1β (diluted 1 : 500, ab2105, Abcam), anti-IL-1β (diluted 1 : 1500, ab9722, Abcam), anti-p-NF-κB (p-p65) (diluted 1 : 2000, ab86299, Abcam), anti-NF-kB p65 (diluted 1 : 300, ab19870, Abcam), anti-TLR4 (diluted 1 : 500, ab13556, Abcam), anti-MyD88 (diluted 1 : 1000, ab133739, Abcam), and anti-β-actin (diluted 1 : 1000, ab8226, Abcam). β-Actin was used as an internal control.

Samples from patients with PDR or donor eyecups were fixed with freshly prepared paraformaldehyde (4%) for 2 h at 4 °C, dehydrated using 30% sucrose solutions for 30 min and sent immediately to OCT compound (Sakura, PA, USA) for frozen sections. Frozen sections were cut at 10 μm thickness at −20 °C and stored at −80 °C until staining. Sections were stained by NLRP3 (1:500, Abcam, ab4207) or IL-1β (1:500, Abcam, ab2105) with CD31 (1:500, Abcam, ab24590). HRECs were fixed with freshly prepared paraformaldehyde (4%) for 30 min at 4°C. Then, cells and frozen tissue sections were washed 10 min with PBS (three times), and blocked with 1% FBS in PBS for 1 h at room temperature. After a 10-min washing with PBS three times, samples were incubated with NLRP3 or IL-1β overnight at 4 °C in a humidified chamber. After a 10-min washing with PBS three times, cells were incubated with corresponding secondary antibodies conjugated with Alexa Fluor 488 (1:500, Abcam) or Alexa 594 (1:500, Abcam) for 1 h at 37 °C in a darkened humidified chamber. Cell nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI, Sigma, St. Louis, MO, USA). Fluorescence was observed using a confocal microscope (Olympus 1X81, Olympus, Tokyo, Japan).

Mitral valve tissues were fixed in 4% PFA for 24 h. After fixation, the tissue was dehydrated to enable embedding in paraffin, which is water insoluble. The tissue was dehydrated gently by immersion in increasing concentrations of alcohol. The alcohol was then cleared by incubation in xylene prior to paraffin embedding. The paraffin was typically heated to 60 °C and allowed to harden overnight. Finally, the tissue was sectioned into 8-μm-thick paraffin sections using a microtome. Sections were deparaffinized and rehydrated. Endogenous peroxidase activity was blocked by incubation for 30 min in 3% H₂O₂ in methanol at room temperature. Antigen retrieval was performed using microwave treatment for 15 min in citrate buffer (pH 6.0). The sections were blocked with 10% goat serum at 37 °C for 1 h and incubated with rabbit polyclonal antibody against human IL-1β or rabbit monoclonal antibody against human IL1RA (both applied at 1:200, Abcam, USA) in a humidified chamber overnight at 4 °C. Next, the sections were incubated with HRP conjugated secondary antibody for 1 h at room temperature, developed with DAB chromogen for 10 min at room temperature, rinsed in running tap water for 5 min, and counterstained with haematoxylin-eosin staining. The antibodies used in the present study are as follows: anti-IL1β antibody (ab2105, Abcam, USA) and anti-IL1R1 antibody (ab106278, Abcam, USA).

Western blot was completed as previously described by us [5 (link),21 (link),22 (link)]. In brief, the soleus muscle was homogenized using radioimmunoprecipictation assay cell lysis buffer and the supernatant was used. Using the Bio-Rad protein assay (Bio-Rad, Hercules, CA, USA), protein concentration was estimated and 50 µg of protein was run onto a 10% or 15% SDS-PAGE gel (150 V for 1 h). Using a semi-dry transfer machine (Bio-Rad), gels were transblotted onto polyvinylidene difluoride membranes (Bio-Rad) and were subsequently blocked with 5% skim milk. Next, the membranes were incubates with primary antibodies overnight at 4 °C for inflammasome markers including TLR4 (1:1000, Cat# ab13556, 90 kDa; Abcam) and NLRP3 (1:1000, Cat# ab214185, 90 kDa; Abcam). Pyroptosis markers were detected using primary antibodies for IL-1β (1:1000, Cat# ab2105, 31 kDa; Abcam), and IL-18 (1:1000, Cat# ab7149, 22 kDa; Abcam). Inflammation was detected by primary antibody for TNF-α at a 1:500 dilution (Cat# ab6671, 17–22 kDa; Abcam). Finally, primary antibody for β-actin was used as a loading control at a 1:1000 dilution (Cat# ab16039, 45 kDa; Cell Signaling Technology, Danvers, MA, USA). Membranes were exposed to enhanced chemiluminescence substrate (ThermoFisher Scientific). Densitometric analysis was performed using NIH ImageJ software.