Rabbit anti nox4

Western blotting was performed as previously described [26 ]. Immediately after enucleation, retinas were dissected and lysed in 1X Mg2+ lysis buffer (EMD Millipore) with a protease inhibitor (Sigma Aldrich, St. Louis, MO) and a phosphatase inhibitor (EMD Millipore). Samples were quantified with Bradford Reagent (Bio-Rad) [27 (link)]. 40 μg of total protein were separated on 4–20% Tris-glycine gel (Bio-Rad), transferred to nitrocellulose membrane (Bio-Rad), and blocked with 5% BSA diluted in Tris-buffered saline supplemented with Tween-20 (TBST) for one hour. Membranes were incubated with rabbit anti-NOX4 (1:200 dilution, sc-30141, Santa Cruz, Dallas, TX) for 18 hours or mouse anti-β-actin (1:5000 dilution, A5316, Sigma-Aldrich) diluted in 5% BSA in TBST for one hour. After washing, membranes were incubated with donkey anti-rabbit (1:5000 dilution, NA934, GE Healthcare, Piscataway, NJ) or horse anti-mouse IgG (1:5000 dilution, 7076, Cell Signaling, Boston, MA) and exposed with ECL Detection Reagents (GE Healthcare). Bands were analyzed using ChemiDoc XRS (Bio-Rad) and normalized to β-actin. Data are fold change ± SEM.

Rat glomerular mesangial cells (RMC, HBZY-1, Life-Science Academy of Wuhan University, Wuhan, China) were cultured and maintained in DMEM (Invitrogen, Carlsbad, CA), PH7.4, supplemented with 10% fetal bovine serum (FBS, Invitrogen, Carlsbad, CA), 2 mM glutamine, 100 U/ml penicillin, 100 µg/ml streptomycin at 37 °C. To examine the effect of compound 1, RMC cells were pre-treated with indicated concentration of compound 1 at 37 °C for 1 h, and then exposed to either 5.6 mM (normal glucose, NG) or 25 mM (high glucose, HG) D-glucose for 24 h or 48 h. Whole cell proteins were extracted using a cell lysis buffer kit (Cell signaling, MA, USA) and quantified by the Bradford assay (Bio-Rad, Hercules, CA). The equivalent amount of proteins were resolved with SDS-PAGE and transferred to nitrocellulose membranes. The membranes were blocked and then incubating with a rabbit anti-fibronectin pAb (Sigma, MO, USA), rabbit anti-collagen I (Abcam, MA, USA), rabbit anti-p47phox, rabbit anti-Nox4, rabbit anti-PCNA, rabbit anti-c-myc (all from Santa Cruz, Texas, USA) and mouse anti-α-tubulin (Sigma) at 4 °C overnight. After washing, the membranes were incubated with HRP-conjugated anti-rabbit or anti-mouse IgG secondary antibodies and detected by using ECL detection system.