Gtx37695

Skin and keratinocytes were solubilized using lysis buffer [1% NP40, 20 mM Tris-HCl (pH 7.5), 0.15 M NaCl, 1 mM EDTA, plus phosphatase cocktails and protease inhibitor cocktails (Nacalai Tesque)]. Each sample was resolved using 10% SDS-polyacrylamide gels, transferred to PVDF membranes, and incubated overnight with anti-phospho-EGFR (Tyr1068) (D7A5) XP^® rabbit monoclonal antibody (dilution ratio 1:1,000) (#3777, Cell Signaling), anti-EGFR (D38B1) XP® rabbit monoclonal antibody (dilution ratio 1:1,000) (#4267, Cell Signaling), anti-ERK1/2 (137F5) rabbit monoclonal antibody (dilution ratio 1:1,000) (#4695 S, Cell Signaling), anti-phospho-ERK1/2 (T202/Y204) (20G11) rabbit monoclonal antibody (dilution ratio 1:1,000) (#4376 S, Cell Signaling), anti-STAT3 rabbit polyclonal antibody (dilution ratio 1:1,000) (#9132 S, Cell Signaling), anti-phospho-STAT3 (Y705) rabbit polyclonal antibody (dilution ratio 1:1,000) (#9131 S, Cell Signaling), and anti-filaggrin antibody (dilution ratio 1:1,000) (#GTX37695, GeneTex). The bound antibodies were detected with anti-rabbit IgG conjugated to horseradish peroxidase (GE Healthcare). The full uncropped blot images are shown in Supplementary Fig. 1.

To investigate the effect of the GTB1 on AD, the lesion area of the test animal tissue was fixed in 10% neutral buffered formalin at the end of the experiment. After step-by-step dehydration using a high-concentration ethanol solution (starting from a low concentration), paraffin blocks were prepared. Each tissue block was cut to a thickness of 5 μm to facilitate attachment to the slide, and each tissue slide was deparaffinized with xylene and dehydrated using alcohol. To evaluate epidermal thickening, tissue slides were stained with hematoxylin and eosin (H&E). Mast cells in the skin were stained with toluidine blue (TB). Some skin sections were stained for anti-filaggrin (1 : 250, GTX37695, Gene Tex) and anti-loricrin (1 : 2000, ab85679, Abcam) antibodies in accordance with the manufacturer's instructions. The slices were washed with PBS, dehydrated, and mounted in a Permount mounting medium (SP15-100, Thermo Scientific). All stained tissue slides were photographed using a slide scanner (Pannoramic MIDI; 3DHISTECH Ltd, Budapest, Hungary) and observed using Case Viewer software. All histological examinations were analyzed in 3 sections/animal slices.