Anti nox4

Band intensities for NOX−4 in the cell lysates were faint to quantify using Western blot analysis. To enhance the signal intensity, cell lysates (500 μg; prepared in RIPA buffer) were immunoprecipitated using 6 μg of anti-NOX−4 (Santa Cruz) antibodies [12 (link)]. The immunoprecipitates were analyzed by Western blot using anti-NOX−4 antibodies (Santa Cruz).

After various treatment, HSCs were washed twice with PBS and scraped in a lysis buffer (20 mM Tris-HCl, 250 mM sucrose, pH 7.2) containing 1X protease inhibitor mixture (Roche, Indianapolis, IN). Proteins were denatured with SDS buffer and boiled for 5 min. Samples were run by SDS-PAGE, transferred onto a PVDF membrane, and blocked using 5% nonfat dry milk (NFDM) in Tris-buffered saline with 0.1% Tween 20 (TBST). Then, the membranes were probed with the following primary antibodies overnight at 4 °C: anti-α-SMA (1:1000, Abcam, Cambridge, MA), anti-desmin (1:500, BD Pharmingen, San Jose, CA), anti-CB1 (1:1000, Thermo, Waltham, MA), anti-CB2 (1:1000, Abcam), anti-TIMP-1 (1:500, Santa Cruz, Dallas, TX), anti-collagen I (1:1000, Calbiochem, Billerica, MA), anti-Nox1 (1:500, Abcam), anti-Nox4 (1:500, Santa Cruz), anti-Nrf2 (1:1000, Santa Cruz), and anti-β-actin (1:10000, Santa Cruz). After washing with TBST three times, membranes were incubated with the following secondary antibodies for 1 h at room temperature, respectively with corresponding goat anti-rabbit-HRP or rabbit anti-mouse-HRP (1:10000, Santa Cruz) based on animals used for raising primary antibody. After washing for the final three times, bands were detected by the addition of a chemiluminescent substrate and visualized on Kodak Omat X-ray film.