Primary antibodies for nrf2

The HaCaT cell line was obtained from the laboratory of Wonkwang university (Prof. Min Cheol Park). The HaCaT cells were incubated in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 100 units/ml of penicillin, and streptomycin (Welgene, Seoul, Korea). The HaCaT cells were cultured at 37°C in an incubator with a humidified atmosphere of 5% CO₂ and 95% air. DMEM and FBS were purchased from GIBCO BRL (Grand Island, NY, USA). penicillin and streptomycin were purchased from Welgene (Seoul, Korea). Recombinant human TNF-α, IFN-γ, and IgE mouse ELISA kit were obtained from BioLegend (San Diego, CA, USA). Primary antibodies for Nrf2, Lamin B, HO-1, β-actin, and secondary antibodies used in the western blot analysis were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). 3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT), dexamethasone (#D2915), and DNCB were purchased from Sigma-Aldrich. (St. Louis, Mo., USA).

To test the effect of C. molmol on Nrf2 and HO-1 expression in the cerebrum, Western blotting was used as we previously reported [48 (link)]. In brief, samples from the cerebrum were homogenized in RIPA buffer with proteinase inhibitors and centrifuged, and protein concentration was determined in the homogenates using Bradford reagent [49 (link)]. To determine Nrf2, nuclear proteins were extracted using a commercial kit purchased from Beyotime (China). The samples were electrophoresed on SDS/PAGE, transferred to PVDF membranes, blocked, and incubated with primary antibodies for Nrf2, lamin B, HO-1, and β-actin (Santa Cruz Biotechnology, USA). After washing, the membranes were incubated with the secondary antibodies, washed, and then developed using enhanced chemiluminescence kit (Bio-Rad, USA). The intensity of bands was determined and quantified using ImageJ (version 1.32j, NIH, USA).