Chemiluminescence method

Tissues or cells were lysed in ice-cold radioimmunoprecipitation assay buffer. The bicinchoninic acid assay (BCA) protein kit (Yesen, Shanghai, China) was used to quantify protein concentration. After loading samples on 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gels, the resolved proteins were transferred onto nitrocellulose membranes (Millipore, Massachusetts, USA). The membranes were probed for 12 h at 4°C using antibodies against Col-1, α-SMA, CD47, E-cadherin, and β-actin, and then probed with secondary antibodies (Zsbio, Beijing, China) for 1.5 h at 37°C. After washing with Tween, the blots were developed using a chemiluminescence method (Thermo Scientific, Waltham, MA, USA). The results were quantified using ImageJ 1.45s software (NIH, Bethesda, MD, USA).

Total protein of skin samples, cell culture lysates and precipitated supernatants was prepared using T-PER Tissue Protein extraction lysis buffer (Thermo Fisher Scientific, Bonn, Germany) and quantified by BCA Protein Assay kit (Thermo Fisher Scientific, Bonn, Germany). Equivalent amounts of proteins (10–15 μg) were separated using a 12% SDS-polyacrylamid gel (Invitrogen, Carlsbad, CA, USA), transferred to reinforced nitrocellulose membranes and blocked (Tris buffered saline, pH 7.5, 0.1% Tween 20 (TBS-T), 5% milk powder) for 1 hour at room temperature. Membranes were incubated with anti-caspase-1 IgG (1:1000), anti-caspase-5 IgG (all Cell Signaling Technology, Inc., Beverly, MA, USA; 1:1000), and anti-NLRP1(Nalpy1-4) IgG (Enzo Life Sciences Inc., Farmingdale, NY, USA; 1:1000) in 5% BSA/TBS-T at 4°C overnight. Gel loading was controlled by detecting β- actin signal with monoclonal antibodies (Cell Signaling, Technology, Inc., Beverly, MA, USA; 1:2000). After washing with TBS-T, blots were developed by incubation with horseradish-peroxidase-conjugated secondary antibodies (Cell Signaling Technology, Inc., Beverly, MA, USA; 1:10.000) for 1 h at room temperature and visualized with chemiluminescence method following the manufacturer’s protocol (Thermo Fisher Scientific, Bonn, Germany).