Anti human cornifelin

Cytotoxicity of Lawsone was measured by phosphorylation at Ser139 residues of the H2A.X histone. Phosphorylation of the H2A.X histone occurs at the site of DNA damage after exposure to polyaromatic hydrocarbons, hydroxyl radicals or ionizing radiation⁶⁹ . 4 h after Lawsone exposure, HEK cells were fixed with 2% paraformaldeyde for 20 min at room temperature (RT) and permeabilized with 0.1% Triton for 5 min at RT. After 30 minutes in blocking buffer, cells were stained with α-phospho-Histone H2A.X (Millipore) for 1 h at RT, followed by staining with the α-rabbit IgG AlexaFluor488 (Dianova,) for 1 h at RT. Nuclei were stained using Nuc red Live 647 (Life technologies). Cell image acquisition and analysis was perfomed using Arrayscan XTI Live High Content Platform (ThermoFisher Scientific).
Formalin-fixed paraffin-embedded skin equivalents were stained either with hematoxylin and eosin (Sigma-Aldrich) or anti-human cornifelin (Sigma) and loricrin (Abcam), followed by anti-rabbit AlexaFluor555 and 488 respectively. Nuclei were counterstained with DAPI. Images were acquired using a Leica DMRB fluorescent microscope and analyzed with ImageJ (https://imagej.nih.gov/ij/).

Proteins of human skin equivalents were isolated with radioimmunoprecipitation assay buffer and protein concentrations analyzed with the Pierce BCA Protein Assay Kit (Thermo Fisher Scientific), according to the manufacturer’s instructions. Anti-human cornifelin and filaggrin were purchased from Sigma and anti-human loricrin from Abcam.
AhR protein in HEK was detected after cell lysis with RIPA buffer. Protein amount was quantified using Pierce BCA Protein Assay Kit (Termo Fisher Scientific), according to the manufacturer’s instructions. 30 μg protein were diluted in Laemmli buffer containing β-mercaptoethanol and loaded on a Mini Protean TGX Stain Free precast Gel. AhR protein was detected by ECL, using anti-AhR polyclonal antibody. (Enzo Life sciences) and β-Actin expression (Abcam) was used as a loading control.