Nrf 2 antibodies

Total proteins were isolated from the lung tissue homogenates according to the protocol provided by the Protein Extraction Reagents Kit (ASPEN, South Africa). Proteins were separated on 10% SDS-PAGE gels by electrophoresis and transferred to a PVDF membrane. The membranes were blocked with 5% nonfat milk for 1 hour and probed with the following antibodies: Nrf-2 antibodies (1 : 200, Santa Cruz, California, USA), HO-1 antibodies (1 : 200, Santa Cruz, California, USA), H2A antibodies (1 : 1000, Santa Cruz, California, USA), and β-actin antibodies (1 : 2000, Merck Millipore, Germany). HP-conjugated anti-mouse IgG (Cell Signaling Technology) was used as a secondary antibody. Images were scanned with the canon imaging system and analyzed using Alpha image software.

ChIP and qPCR analysis were performed using an EZ-ChIP assay kit (17-295, EMD Millipore, Burlington, VT, USA). Briefly, BMDMΦs or MH-S cells treated with LPS were crosslinked with formaldehyde, and chromatin was prepared according the protocol. Diluted and pre-cleared chromatin was incubated with Nrf2 antibodies (SC-365949, Santa Cruz Biotechnology, Inc., Dallas, TX, USA), IgG or RNA Pol II antibodies (17-295, EMD Millipore) for 18 h at 4 °C with rotation. Samples were incubated with protein A-agarose, washed and bound DNA was eluted. Immunoprecipitated DNA was amplified by qPCR using murine A20 promoter (Accession KX869907) specific primers: Forward, 5′-CTATTTGCTGCCTTGTAGCATC-3′ and Reverse, 5′-GTTTGTCAGTGATCCAAGTTTGT-3.’