Rabbit anti p47phox

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.

Samples, including whole lysates, and cytosolic and membrane fractions from cells and tissues were subjected to Western blot analysis. A 30 μg protein sample was separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis; electrotransferred to PVDF membranes; blocked with 5% BSA in TBST; and then immunoblotted with rabbit anti-p47phox (1 : 1000, Santa Cruz, USA), rabbit anti-cleaved caspase-3 (1 : 1000, CST, Danvers, Massachusetts, USA), rabbit anti-γ-H2Ax (1 : 1000, CST, Danvers, Massachusetts, USA), mouse anti-pan-cadherin (1 : 1000, Abcam, UK), or rabbit anti-β-tubulin antibody (1 : 4000, Bioworld, China) diluted in blocking solution containing 5% BSA and 0.1% Tween-20 in Tris-HCl-buffered saline overnight at 4°C. After being rinsed, membranes were incubated with HRP-conjugated anti-rabbit immunoglobulin or anti-mouse immunoglobulin at 1 : 5000 for 1 hour. Specific proteins were detected by enhanced chemiluminescence. The membranes were then exposed to X-ray film. The densities of bands were measured using a densitometer and analyzed with ImageJ. All the target protein expressions were normalized to their corresponding β-tubulin or pan-cadherin products.