Bs 2013r

Tissue samples were homogenized in lysis buffer (50 mM of Tris–HCl, 150 mM of NaCl, 2 mM of EDTA, 2 mM of EGTA, 0.2% Triton X-100, 0.3% NP-40, 0.1 mM of PMSF, and 1 µg/ml of pepstatin A) and then separated by 10% SDS-PAGE under reducing conditions. After electrophoresis, samples were transferred to PVDF membranes (IPVH00010, Millipore, Bedford, MA, USA), blocked with 5% skimmed milk in TBS/0.5% v/v Tween 20 for 1 h, washed with TBS/Tween, and incubated with anti-HO-1 (1:2000 dilution, ADI-OSA-150-D Enzo Life Technologies), rabbit anti-ferritin light chain (1:500 dilution, ab69090, Abcam), rabbit anti-ferritin heavy chain (1:1000 dilution, Thermo Fisher 701934), and anti-phospho Nrf2 (1:1000 dilution, bs-2013R, Bioss). Antibodies were diluted in 5% milk TBS/Tween. Blots were washed with TBS/Tween and incubated with anti-rabbit horseradish peroxidase-conjugated secondary antibody (1:2000, Amersham, Aylesbury, UK). After being washed with TBS/Tween, blots were developed with the chemiluminescence method (ECL Luminata Crescendo, WBLUR0500, Millipore) and scanned using the ImageQuant LAS-4000 (GE Healthcare). Blots were then probed with mouse monoclonal anti-α-tubulin antibody (1:5000, T6199, Sigma, MO, USA), and levels of expression were corrected for minor differences in loading. Quantification was expressed as arbitrary densitometric units (AU).

Human skin biopsy specimens and mouse ear specimens were fixed in formalin (10%) and embedded in paraffin. 8 μm sections were cut on a microtome in the same orientation. Skin architecture, melanin deposition, and melanocytes were assessed following H&E and Fontana-Masson (F&M) staining, respectively. Tissue was also incubated with primary antibodies and Alexa Fluor–conjugated secondary antibodies for indirect IF. The primary antibodies used in this study included rabbit polyclonal antibodies against NRF2 (sc-13032; Santa Cruz Biotechnology), P-NRF2 (bs-2013R; Bioss Antibodies), and HO-1 (NB110-57028; Novus Biologicals). Immunohistochemistry for tyrosinase expression was performed by the Johns Hopkins Reference Histology and Immunopathology Core, according to guidelines for clinical specimens. Bright-field and IF microscopy images were obtained using a Leica DFC495 microscope (Leica).