Anti involucrin

The 4% formaldehyde-fixed and dehydrated skin tissue samples were embedded in paraffin wax and cut into 5-µm-thick sections. The skin sections were incubated with a primary antibody against human KRT10 (1:100; Abcam, Cambridge, UK), followed by incubation with the adequate secondary antibody. After staining, sections were counterstained with hematoxylin to provide contrast. For immunofluorescence staining, the skin tissues were quench-frozen and embedded in OCT. The cryosections (10 µm) were fixed in chilled acetone for 15 min and incubated overnight at 4 °C with anti-S100A9 (1:400; Abcam, Cambridge, UK), anti-HBD-2 (1:400; Abcam, Cambridge, UK), anti-Filaggrin (1:400; Abcam, Cambridge, UK), anti-Involucrin (1:400; Abcam, Cambridge, UK), anti-AO-1 (1:400; Abcam, Cambridge, UK), anti-CLDN-1 (1:400; Abcam, Cambridge, UK), anti-KRT6 (1:400; Abcam, Cambridge, UK), anti-KRT10 (1:400; Abcam, Cambridge, UK), and anti-KRT17 (1:400; Abcam, Cambridge, UK) primary antibodies, followed by Alexa Fluor 488- or 568-conjugated secondary antibodies and DAPI. The immunostained images were obtained using a digital camera (DP74; Olympus, Tokyo, Japan) coupled with an optical microscope (BX53; Olympus, Tokyo, Japan).

The WB procedure was performed as described previously
[39 (link)] with some modifications. Briefly, after transient transfection with plasmids, cells were harvested and lysed in a lysis buffer containing a cocktail of protease inhibitors. After centrifugation at 12,000 rpm for 15 min at 4°C, supernatants were collected, mixed with dithiothreitol, and used for WB. The ProteoExtract® Subcellular Proteome Extraction Kit (Millipore) was used for extraction of subcellular fractions following the manufacturer’s protocol. Equal amounts of protein extract were electrophoresed in 10% SDS-PAGE gels and then transferred to nitrocellulose membranes. The membranes were blocked with 5% bovine serum albumin (BSA) at room temperature (RT) for 1 h, incubated with the primary antibody overnight at 4°C, and incubated with the secondary antibody at room temperature for 1 h. Blots were washed in Tris-buffered saline with 0.1% Tween-20 and proteins were visualized by chemiluminescence. The following antibodies were used for WB: anti-SOX1 (IB 1:2000, Epitomics, CA, USA), anti-CK19 (1:8000, Epitomics), anti-CK18 (1:4000, Epitomics), anti-CK13 (1:4000, Epitomics), anti-CK8 (1:4000, Epitomics), anti-Involucrin (1:4000, Abcam, MA, USA), anti-GAPDH (1:10000, ProteinTec Group, IL, USA), anti-c-Myc (N-262) (1:2000, Santa Cruz Biotechnology, CA, USA), and anti-β-catenin (1:2000, Upstate, NY, USA).

Cells and dorsal tissues were collected and lysed in PRO-PREP (iNtRON, Seongnam, Korea) containing a protease inhibitor cocktail (Complete™; Roche, Mannheim, Germany). Amounts of protein in the lysates were quantitated using a BCA kit (Thermo Fisher Scientific). Following quantitation, equal amounts of protein were resolved on 10% SDS-PAGE gels and electrotransferred to polyvinylidene fluoride membranes (Millipore, Bedford, MA, USA). After blocking with 5% skim milk in Tris-buffered saline containing 0.5% Tween-20 (TBST), membranes were probed with anti-filaggrin, anti-involucrin, anti-loricrin from Abcam (Cambridge, UK); anti-alix, anti-flotillin, anti-CD9, anti-phospho-STAT1, anti-STAT1, anti-phospho-STAT1, anti-STAT1, anti-phospho-STAT3, anti-STAT3 from Cell Singling Technology (Danvers, MA, USA); and anti-β-actin (Santa Cruz Biotechnology, Santa Cruz, CA, USA) antibodies at 4 °C overnight. After washing, membranes were incubated with HRP-conjugated anti-mouse (Vector Labs Inc., Burlingame, CA, USA) or anti-rabbit (Vector Labs Inc.) secondary antibodies. Immunoreactive signals were detected using enhanced chemiluminescence (ECL) reagents (EzWestLumi plus; ATTO, Tokyo, Japan).