Anti nox1

Western blotting analyses were performed as described previously (Demelash et al. 2015). The membranes were visualized using enhanced chemiluminescence (ECL) reagents (GE Healthcare) or WesternBright Quantum kit (Advansta, Menlo Park, California, USA). The following antibodies (at the indicated dilutions) were used in this study: anti-NOX1 (rabbit, 1:500, Santa Cruz Biotechnology); anti-NOX4 (rabbit, 1:1000, Santa Cruz Biotechnology); anti-Mcl-1 (rabbit, 1:1000); anti-phospho-ATM Ser198 (ATM-S1981^p, rabbit, 1:500); anti-phospho-ATR Ser428^p (ATR-S rabbit, 1:500); anti-phospho-Chk1Ser345^p (rabbit, 1:500); anti-phospho-Chk2T68^p (rabbit 1:500). Antibodies were purchased from Cell Signaling Technology. Mouse anti-β-actin (Santa Cruz Biotechnology) at a dilution of 1:10000 was used as loading control.

Cells were lysed using RIPA lysis buffer (T&I, Chuncheon, Kangwon, Korea) supplemented with a phosphatase and protease inhibitor cocktail (Roche, Grenzacherstrasse, Basel, Switzerland), and the lysates were centrifuged at 13,000 rpm for 15 min at 4 °C to separate the supernatants. Cell lysates were subjected to SDS-PAGE gels and transferred to PVDF membranes (Merck Millipore, Burlington, MA, USA). The membranes were blocked for 1 h with 5% skim milk (BD Biosciences, Franklin Lakes, NJ, USA), and then the primary antibodies were added and incubated overnight at 4 °C. The blots were detected using anti-phospho-PAK1 (1:1000, Cell signaling, Danvers, MA, USA), anti-PAK1 (1:1000, Santa Cruz, Santa Cruz, CA, USA), anti-phospho-AKT (1:1000, Cell signaling, Danvers, MA, USA), anti-AKT (1:1000, Cell Signaling, Danvers, MA, USA), anti-NOX1 (1:1000, Santa Cruz, Santa Cruz, CA, USA), and anti-GAPDH (1:5000, Santa Cruz, Santa Cruz, CA, USA) antibodies. The specific protein bands were detected using chemiluminescence reagents (GE Healthcare Life Science, Chicago, IL, USA) and quantified using ImageJ software (National Institutes of Health, Bethesda, MD, USA).