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Materials and methods
Materials. Following chemicals were used; cytochrome c (Wako), SOD (Wako), PMA (Sigma) and DPI (Sigma). Following GSK690693 were purchased; CD3 (BioLegend), F4/80 and CD45R (eBioscience), 7/4 (Serotec), p22, p47 and p67 (Santa Cruz Biotechnology). VSOP antibody is the polyclonal antibody to the C-terminal cytoplasmic region of mouse VSOP/Hv1 that has previously been described [18]. Mac1 antibody was provided from Developmental Studies Hybridoma Bank. gp91 antibody was provided by Dr. Mark Quinn.
Generation of VSOP/Hv1 gene-trap mouse. Embryonic stem cell line RPN293 with a disrupted VSOP gene was purchased from Bay Genomics. In the embryonic stem cells, the gene-trap vector pGT0Lxf, which is 8.5 kb in length and is composed of a splice-acceptor site followed by a ‘beta-Geo’ cassette encoding a fusion of beta-galactosidase and neomycin phosphotransferase, was inserted in the intron between exon 3 and exon 4 of VSOP gene ( Supplementary. Fig. 1A). Chimeric mice were made at the laboratory animal resource center at University of Tsukuba. Chimeric mice were backcrossed against C57BL/6J for eight generations. Animal experiment was approved by Animal Research Committees of Graduate School of Medicine, Osaka University and National Institutes of Natural Sciences.