8 oxo dg antibody

Immunofluorescence staining was performed following the manufacturer’s protocol (Trevigen, Gaithersburg, MD) with some modifications described as follows. 6×10⁴ AsPC-1 cells were seeded onto 4-well glass chamber slides, then treated with either solvent or IL-4 (50 ng/ml) plus IL-17A (50 ng/ml) for 48 h. The primary antibodies were incubated overnight at 37ºC on a humidified chamber with dilution 1:2000 in 1X PBS containing 1% BSA and 0.01% Tween 20. The secondary antibodies were incubated using a dilution of 1:400 in 1X PBS containing 1 % BSA for 1 h at room temperature in the dark. Slides were mounted using VECTASHIELD antifade mounting medium (cat # H-1200, Vector Laboratories, Burlingame, CA) with DAPI for counterstaining of nuclei. The antibodies for immunofluorescence staining of 8-oxo-dG were a mouse monoclonal 8-oxo-dG antibody, cat# 4354-MC-050 from Trevigen, Gaithersburg, MD; and a goat anti-mouse secondary antibody conjugated with Alexa Fluor 488, cat# A-11001 from Thermo Scientific, Rockford, IL. Fluorescence images were captured using a confocal microscope (Zeiss LSM 780); Zen Blue version 2.3 software was used for image quantitation.

Bixin was purchased from Spectrum (New Brunswick, NJ); sodium arsenite (As), cycloheximide, and MG132 were from Sigma (St. Louis, MO); sulforaphane (SF) was purchased from Santa Cruz (Santa Cruz, CA); primary antibodies against NRF2, KEAP1, GCLM, AKR1C1, NQO1, AKR1C2, HO-1, TrxR, FTH (heavy), MMP9, Ki67, and GAPDH, as well as horseradish peroxidase (HRP)-conjugated secondary antibodies were purchased from Santa Cruz. Antibodies against p-P65 and P65 were purchased from Cell Signaling. The anti-Thymine Dimer (H3) CPD antibody was purchased from Novus (Littleton, CO). The hemagglutinin (HA) epitope antibody was purchased from Covance (Branford, CT). The 8-oxo-dG antibody was purchased from Trevigen (Gaithersburg, MD). Human immortalized HaCaT keratinocytes were grown in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum and 0.1% gentamycin, and primary human epidermal keratinocytes [adult HEKa (C-005-5C)] were cultured on collagen matrix protein-coated dishes using Epilife medium (EDGS growth supplement; Life Technologies, Carlsbad, CA).