Novex wedgewell 4 12 tris glycine mini gels

ABCB5+ DSCs, fibroblasts, keratinocytes and BM-MSCs were either lysed on ice for 10 minutes in RIPA Lysis Buffer for whole cell lysate (Santa Cruz Biotechnology) or cell lysis solution for extracellular matrix (ECM) protein extraction (5ml 1M NH4OH + 1.25ml Triton-100 + H₂O to 250 mLs) at room temperature for one minute. ECM proteins were then dissolved in 4% SDS in 0.1M Tris·HCl. Equal quantities of protein (40 μg) were loaded onto a Novex WedgeWell 4–12% Tris-Glycine Mini Gels (Thermo Fisher). Following electrophoresis, protein was transferred onto a nitrocellulose membrane and incubated with anti-ABCB5 antibody (bs-1604R; Bioss), anti-C7antibody (sc-20774; Santa Cruz or gift from Dr. Woodley), anti-laminin alpha-3 (ab242197; Abcam), anti-laminin 332 (ab78286, Abcam), anti-laminin beta-3 (OTI3A2; Bio-rad) or anti-beta-actin antibody (A2547; Millipore Sigma) at 4°C overnight. Membranes were then incubated with goat anti-rabbit HRP-conjugated secondary antibody (sc-2004; Santa Cruz Biotechnology) or anti-mouse HRP-conjugated secondary antibody (sc-2005; Santa Cruz Biotechnology). Blots were developed using SuperSignal West Femto Chemiluminescent Substrate (Thermo Fisher) for C7, laminin alpha-3, laminin 332 and laminin beta-3 or using SuperSignal West Pico Chemiluminescent Substrate (Thermo Fisher) for beta-actin and developed on X-ray film.

Histone protein was extracted with the Histone Extraction Kit (Abcam), according to the manufacturer’s protocol. The protein samples were denatured and separated by Novex^™ WedgeWell^™ 4–12% Tris-Glycine Mini Gels (Thermo Fisher Scientific) and transferred onto 0.2 μm PVDF Pre-cut Blotting Membranes (Thermo Fisher Scientific). After blocking with 5% non-fat milk at room temperature for 2 hours, the membranes were incubated with primary antibodies to H3K36me2 or H3K27me3 or total H3 at 4 deg C overnight. Antibody details are in Supp. Table 1. Subsequently, the membranes were incubated with secondary antibodies for 1 hour at room temperature. Finally, protein signals were detected by Pierce^™ ECL Western Blotting Substrate (Thermo Fisher Scientific) and visualized after exposure to the Hyperfilm^™ ECL^™ (GE Healthcare). The relative optical density ratio was calculated with the Image J software (RRID:SCR_003070) by comparison to H3. Original Western blot gel images and Image J data are provided in Supp. Data 2 and Supp. Data 3.