Anti filaggrin

Dorsal mouse skins were collected and treated with trypsin overnight for epidermis and dermis separation. Epidermis was sonicated for lysis in denaturing buffer composed of 300 mM NaCl, 2 mM EDTA, 20 mM Hepes, 1% SDS,0.1 mM hemin chloride, 5 mM NEM, 20 mM NaF, 100mM PMSF. Lysates were centrifuged to remove DNA and cell debris. Protein concentrations were measured by the BCA protein assay (Thermo Scientific Inc., Rockford, IL). 30 ug of lysate protein were loaded for electrophoresis on 4–12% SDS-PAGE gels (Invitrogen, Carlsbad, CA, USA). After electrophoresis, transfer to PVDF membrane (Invitrogen, Carlsbad, CA, USA) and incubating with blocking solution (5% Nonfat Milk) and subsequently with primary antibody was followed. Blots were then rinsed in TBST and incubated in peroxidase-conjugated secondary antibodies, respectively. Proteins were visualized using Bio-Rad Chemi Doc XRS with ECL (Pierce Biotech, Waltham, MA, USA). Antibodies used: anti-DLX3 (Morasso lab; (Hwang et al., 2008 (link)), anti-CD3 (BD Bioscience), anti-CD4 (Abcam), anti-Filaggrin, (Covance), anti-K6 (Covance), anti-K10 (Covance), anti-Ki67 (Biolegend), anti-langerin (Novus Biologicals), anti-pSTAT3 (abcam), anti-STAT3 (Cell Signaling), anti-RPL11 (Bethyl) and anti-Vinculin (abcam).

Antibodies and dilutions used: anti-p53 (Leica Biosystems Novacastra #NCL-p53-CM5p, 1:500 for IHC; Calbiochem #OP29, 5 μg/ml for immunoblots), anti-Grasp (1:3000; previously described in Ref. [1 (link)]), anti-BrdU (1:100, #1370030, AbD Serotec), and anti-β-actin (1:5000, #A5441, Sigma-Aldrich). Antibodies for differentiation markers were purchased from Covance: anti-K14 (1:500; #PRB-155P), anti-K10 (1:500; #PRB-159P), anti-Filaggrin (1:250; #PRB-417P), anti-Loricrin (1:500; #PRB-145P). An anti-Ctip2/Bcl11b antibody (#ab18465 Abcam; 1:2000) was used as a control.