Nox 4 antibody

Forty micrograms of total protein were used for Western blot analysis. Protein expression levels were quantified after immunoblotting using a 1:1,000 dilution of the following specific antibodies: connecting tissue growth factor (CTGF; Cat. No. sc-25440, Santa Cruz Biotechnology), PAI-1 (Cat. No. SC-8979, Santa Cruz Biotechnology), TGF-β (Cat. No. SC-130348, Santa Cruz Biotechnology), COX-2 antibody (Cayman, Ann Arbor, MI, USA), mouse anti-phospho-p44/42 ERK1/2 (Thr202/Tyr204), and a rabbit anti-total ERK antibody (Cell Signaling Technology, Beverly, MA, USA). NOX-4 antibody was purchased from Santa Cruz (sc-21860). Antibodies against Anti-Smad2/3 antibody and anti p-Smad2/3 were obtained from Abcam (Abcam, Cambridge, MA, USA). Primary antibodies were followed by incubation with either donkey anti-rabbit or anti-mouse IgG IRDye 800 CW (Santa Cruz Biotechnology) at 1:3,000 dilutions. Resulting bands were compared to molecular weight standards (M. Biosources, San Diego, CA, USA). Densitometry was performed with ImageJ software and normalized to monoclonal anti-β-actin antibody (Cat. A2228, Sigma Chemical Co, St. Louis, MO, USA).

To determine Nox4 expression, 24 h after treatment, the cells were affixed with paraformaldehyde (4%) in PBS at room temperature for 15 min before being permeabilized with 0.1% Triton X-100/0.1% sodium citrate for 20 min. The cells were blocked with bovine serum albumin (5%) and incubated with Nox4 antibody (1:200; sc-518092, Santa Cruz Biotechnology, Dallas, TX, United States) diluted in 1% skimmed milk solution overnight. Then, the cells were incubated with Alexa Fluor 488 goat anti-mouse antibody (1:200; A-11059, Invitrogen, Waltham, MA, USA) for 1 h and rinsed with PBS. The cellular nuclei were stained blue with 4′,6-diamidino-2-phenylindole (DAPI; ab228549, Abcam, Cambridge, MA, USA) and analyzed under a fluorescence microscope (Olympus Optical, Tokyo, Japan).