Nox2 sirna

Bromodichloromethane (BDCM), a known substrate for CYP2E1-mediated oxidative stress in the liver and corn oil were purchased from Sigma-Aldrich (St. Louis, MO). Anti–α-SMA, anti–IL-1β, anti–TNF-α, anti-TLR-4, Anti-3-Nitrotyrosine (3-NT), anti-P47phox and anti-GP91phox primary antibodies were purchased from Abcam (Cambridge, MA). Species-specific biotinylated conjugated secondary antibody and streptavidin -HRP were purchased from Vectastain Elite ABC kit (Vector Laboratories, Burlingame, CA). Wild-type and gene-specific KO mice were purchased from The Jackson Laboratories (Bar Harbor, ME). NOX2-siRNA, RNAiMAX-Lipofectamine, Fluorescence-conjugated (Alexa fluor) secondary antibodies and ProLong Gold antifade mounting media with DAPI were bought from Thermo Fisher Scientific (Waltham, MA). Animal diets were purchased from Research Diets (New Brunswick, NJ). All other chemicals were purchased from Sigma Aldrich unless otherwise specified. Paraffinized tissue sections on slides were done by IRF, University of South Carolina School of Medicine and AML laboratories (Baltimore, MD).

To introduce siRNA into rat DRG neurons, the cells were plated on 6-well plates at 90% confluence before transfection. Individual siRNAs (at 5 nM), lipofectamine 3000 and Opti-MEM were mixed and incubated at room temperature for 20 min siRNA– lipofectamine 3000 complexes were added to cells for 24 h and the medium was replaced by fresh serum DMEM medium after transfection. Nox1 siRNA (190372), Nox2 siRNA (189525) and Nox4 siRNA (190243) were purchased from Thermo Fisher Scientific (MA, USA). Experiments were performed 72 h after transfection. Knockdown of Nox1, Nox2 or Nox4 was assessed by western blots assay [32] (link).