Protein inhibitor cocktail

Epidermal cell keratinocytes were isolated from WT and ADAM10^ΔMx1 mice 30 days post-induction with poly(I:C) via CD45 MicroBeads and auto MACS pro. Protein lysates were prepared using RIPA buffer (Cell Signaling Technology) supplemented with protein inhibitor cocktail (Cell Signaling Technology). Following centrifugation, cell lysates were dissolved in 4x sample buffer (Invitrogen) containing NuPAGE sample reducing agent (Invitrogen) and boiled at 95°C for 5min. A total of 20 ug lysates were separated by electrophoresis (4-20% SDS PAGE, Bio-Rad) and transferred on PVDF membranes (Bio-Rad) using Trans-Blot Turbo transfer system (Bio-Rad). Proteins were blotted with a rat anti-Caspase 11/4 antibody (Invitrogen), mouse anti-Caspase 1 antibody (Invitrogen), rabbit anti-GSDMC (Proteintech), rabbit anti-GSDMA (LS Bio), and HRP-conjugated Vinculin (Santa Cruz) antibodies. The anti-GSDMA antibody is expected to cross-react with gasdermin A3 based on sequence homology. Membrane was then incubated with goat anti-rabbit, -mouse or -rat IgG-HRP conjugates (Jackson Immuno Research) secondary antibodies. Following three washing with TBST (Thermo Fisher Scientific), membrane was developed by using Pierce ECL Western blotting substrate (Thermo Fisher Scientific) and images were acquired using a ChemiDoc Touch Image System (Bio-Rad). Protein loading was normalized against vinculin.

Cell layers were harvested in lysis buffer containing 150mM NaCl, 50mM TrisCl, 1mM EDTA, 1% Triton, 1% sodium deoxycholate, 0.1% SDS and protein inhibitor cocktail (Cell Signaling) and frozen at −80°C. Samples were thawed, homogenized on ice and centrifuged at 12,600 rpm for 10 min at 4°C, and protein levels were quantified from the supernatants using the BCA assay (Pierce). 10ug or 25ug of protein (for osteopontin or Runx2 respectively) were mixed with Laemmli loading buffer, boiled and cooled, and separated using SDS-PAGE. Proteins were transferred onto PVDF membranes (Millipore) and blocked in 5% non-fat milk. Membranes were first probed with primary antibodies in 5% non-fat milk or bovine serum albumin and then with horse radish peroxidase (HRP)-conjugated secondary antibodies in 5% non-fat milk. Bands were visualized by chemiluminescence using SuperSignal West Pico substrate (Thermo Scientific). The following primary antibodies and dilutions were used: goat anti-osteopontin (AF808, R&D Systems) at 1:5000, mouse anti-Runx2 (D130-3, MBL International) at 1:500 and rabbit anti-GAPDH (14C10, Cell Signaling Technology) at 1:1000. The following HRP-conjugated secondary antibodies and dilutions were used: anti-goat (A8919, Sigma Aldrich) at 1:20000, anti-mouse (W402B, Promega) at 1:7500 and anti-rabbit (A0545 Sigma Aldrich) at 1:5000.

0.1g of minced skin was homogenized in 1.5 mL eppendorf tube with tungsten carbide beads (Qiagen) and 300ul lysis buffer using TissueLyser LT (Qiagen). Protein lysates were prepared using RIPA buffer (Cell Signaling Technology) supplemented with protein inhibitor cocktail (Cell Signaling Technology). The homogenates were centrifuged at 13,000 rpm for 10 minutes at 4°C. CCL20 production from skin and serum were determined using Mouse MIP3 alpha ELISA Kit (CCL20) (abcam) respectively.