Actin sc 1616 r

DUSP4 (sc-1200), gp91-phox (sc-5827), Nox4 (sc-21860), and actin (sc-1616-R) antibodies were obtained from Santa Cruz (Santa Cruz, CA), p-p38 (4511S), p38 (9212S), p-ERK1/2 (9101S), ERK1/2 (9102S), p-JNK (4671S), JNK (3708S), cleaved caspase-3 (9664S), and MK-2 antibody sampler kit antibodies (9329S) from Cell Signaling (Cambridge, MA). Secondary anti-rabbit (NA934V) and anti-mouse (NXA931) IgG-HRP antibodies were purchased from GE Life Sciences (Piscataway, NJ). p38 inhibitor, SB203580, was purchased from Cayman Chemical (Ann Arbor, MI). Click-iT TUNEL Alexa Fluor 488 Imaging Assay Kit and dihydroethidium (DHE) were purchased from Life Technologies (Grand Island, NY).

Western blot was performed as described previously [24 (link)]. Briefly, after washing with CMF-PBS solution, cells were harvested on ice into lysis buffer containing 1% protease inhibitor and phosphatase inhibitor cocktails (Sigma-Aldrich, St. Louis, MO). Cells were lysed with sonication and cell debris removed by centrifugation at 12,000 RPM for 15 min at 4°C. The supernatant was collected and protein concentration determined using the Pierce BCA assay (ThermoFisher Scientific, Waltham, MA). Proteins (25 µg) were resolved through SDS-PAGE on a 10% Tris-HCl gel and transferred onto nitrocellulose membrane (Bio-Rad Laboratories, Hercules, CA). PC (sc-67021) and actin (sc-1616-R) antibodies were purchased from Santa Cruz Biotechnology Inc. (Santa Cruz, CA). Antigen-antibody complexes were detected using Lumiglo Reagent (Cell Signaling Technology, Inc., Danvers, MA). Density of bands was assessed using UN-SCANIT 6.1 (Silk Scientific Inc., Orem, UT). Band densities were in linear range of detectability and final expression is represented as PC/actin relative to vehicle.