Precast polyacrylamide gel

Keratinocytes were lysed in 6.5 M urea buffer (85 (link)) supplemented with a mixture of phosphatase (Cocktail 2 and 3, Merck) and protease inhibitors (Merck). Proteins (5–15 μg) were run under reducing conditions using 4–12% precast polyacrylamide gels (Thermo Fisher Scientific, Waltham, MA, USA) or 10% polyacrylamide handmade gels, depending on the probed proteins. Nitrocellulose membranes were incubated with antibodies listed in Supplementary Material, Table S2. Detection was performed using Amersham ECL Prime (GE Healthcare Life Sciences, Little Chalfont, UK). Images were acquired with ChemiDoc™ XRS+ System (Bio-Rad, Hercules, CA, USA). Band density was evaluated using Image Lab Software (Bio-Rad). Glyceraldheyde-3-phosphate dehydrogenase (GAPDH) and vinculin (VCL) were used as loading controls.

Whole-cell lysates were prepared by scraping cells in SDS sample buffer (125 mM Tris [pH 6.8], 20% glycerol, 4% SDS). Lysates were sheared with a 25G needle and boiled for 10 min at 95 °C. Protein concentration was determined by standard BCA protein assay (Pierce). Equal amounts of protein were separated on precast polyacrylamide gels (Invitrogen). Immunoblotting was done according to standard methods using IRDye800CW- and IRDye680-labeled secondary antibodies for detection on the Odyssey Infrared imager (LI-COR) or using horseradish peroxidase–conjugated secondary antibodies for detection by enhanced chemiluminescence (Supersignal and Supersignal West Pico Plus; Thermo Scientific). Primary antibodies used were against UBE2D (A615, Boston Biochem, 1:2000 dilution or 4330S, CST, 1:500 dilution), V5 (R960-25, Invitrogen, 1:1000 dilution), HA (MMS-101R, Covance, 1:1000 dilution), FLAG (F3165, Sigma, 1:1000 dilution), RPS20 (ab133776, Abcam, 1:1000 dilution), Histone H3 (ab1791, Abcam, 1:10,000 dilution), CRABP1 (HPA017203, Sigma, 1:1000 dilution), TSPAN8 (A06997-1, SanBio, 1:1000 dilution), GAPDH (PA1-987, ThermoFisher, 1:5000 dilution), HSP90 α/β (sc-7947, Santa Cruz, 1:1000 dilution), β-actin (A5316, Sigma, 1:10,000 dilution), and γ-tubulin (T6557, Sigma, 1:10,000 dilution).

Total protein was isolated from cultured KCs and cellular supernatants using RIPA buffer supplemented with protease and phosphatase inhibitors. Protein concentration was measured by Pierce BCA Protein Assay Kit (Thermo Scientific, Waltham, MA) per manufacturer’s directions. 10-25ug protein was run on 4-12% precast polyacrylamide gels (Invitrogen) and transferred to 0.45µm polyvinylidene difluoride membranes. Membranes were blocked with 5% nonfat dry milk or BSA and incubated overnight at 4°C with primary antibodies (1:1000 dilution; XAF1 #13805; TRAIL #3219; IRF-1 #8478; Phospho-MLKL (Ser358) #91689; MLKL #14993; Cell Signaling Technology) followed by HRP-conjugated goat anti-rabbit IgG (1:10,000 dilution; sc-2301 Santa Cruz, Dallas, TX) or HRP-conjugated mouse anti-rabbit IgG (1:2000 dilution; sc-2357 Santa Cruz). Protein bands were detected by chemiluminescence using SuperSignal West Dura Extended Duration Substrate (Thermo Scientific) and imaged by Omega Lum C (Gel Company, San Francisco, CA). Quantification was completed with ImageJ software relative to β-actin loading control (1:1000 dilution; #4967 Cell Signaling Technology).

To evaluate whether thawing temperature at 4, 25, or 37°C modulates the yield and purity of exosome, western blotting for CD63 as exosomal marker was performed. Frozen serum samples from normal healthy subject were thawed at 4, 25, or 37°C for 30 minutes. After exosome isolation by using ExoQuick Exosome Precipitation Solution, protein extraction was performed with RIPA lysis buffer (Santa Cruz Biotechnology, Paso Robles, CA, USA). The samples were subjected to sodium dodecyl sulphate-polyacrylamide gel electrophoresis. In each lane, 10 μl of each sample was loaded and separated in a precast polyacrylamide gel (Thermo Fisher Scientific, Waltham, MA, USA). Proteins were then electrotransferred onto PVDF membrane (Bio-Rad, Hercules, CA, USA). After blocking the membrane with 5% electrophoresis-grade nonfat milk, primary and secondary antibodies were incubated for 60 min each in a 5% milk solution. Immune complexes were visualized by incubating the membranes with an HRP-conjugated anti-mouse antibody using the ECL detection reagent (GE Healthcare, Little Chalfont, Buckinghamshire, UK). The primary antibody was mouse monoclonal CD63 antibody (Ts63, diluted at 1:250, Thermo Fisher Scientific). The secondary antibody was sheep anti-mouse IgG conjugated to HRP (diluted 1:1000, GE Healthcare).