Anti ahr antibody

Total proteins from LNVs and HDFα cells treated with LNVs (10 and 25 μg/ml) or pre-treated with LNVs and then exposed to UVB irradiation (20 mJ/cm²) for 25 seconds were isolated and analyzed by SDS-PAGE followed by western blotting. Antibodies used in the experiments were as follows: anti-HSP70 antibody (Agrisera, Vännäs, Sweden), anti-AhR antibody (Novus Biologicals, Milano, Italy), anti-Nrf2 antibody (Novus Biologicals, Milano, Italy), and anti-Tubulin antibody (Santa Cruz Biotechnology, Heidelberg Germany). The membranes were incubated with HRP-conjugated secondary antibody (Thermo Fisher Scientific, Cambridge, MA, USA) and the chemiluminescent signal was detected by Chemidoc (Biorad, Milan, Italy).

LNVs were labeled with PKH26 Red Fluorescent Cell Linker Kits (Merck KGaA, Darmstadt, Germany) following the datasheet information. Briefly, LNVs were incubated with PKH26 dye for 15 min at room temperature, washed twice in PBS, and resuspended in the growth medium. The labeled LNVs were incubated with HDFα cells for 4 h at 37°C with 5% CO₂ and at 4°C. After incubation, the cells were fixed with PFA 4%, permeabilized with 0.1% TritonX-100, and stained with Actin Green (Molecular Probes, Life Technologies, Carlsbad, CA, USA) and Hoechst (Molecular Probes, Life Technologies, Carlsbad, CA, USA).
HDFα were treated with LNVs (10 and 25 μg/ml) for 24h, then the cells were fixed with PFA 4%, permeabilized with 0.1% TritonX-100, incubated with anti-AhR antibody (Novus Biologicals, Milano, Italy) or anti-Nrf2 antibody (Novus Biologicals, Milano, Italy) for 1h, then washed and incubated with Goat anti-Rabbit IgG Secondary Antibody, DyLight 594 or 488 (Invitrogen) for 1h. Actin was stained using Actin Green (Molecular Probes, Life Technologies, Carlsbad, CA, USA), and nuclei were stained with Hoechst (Molecular Probes, Life Technologies, Carlsbad, CA, USA).
The samples were analyzed by confocal microscopy (Nikon A1, Amsterdam, Netherlands).