Anti nad p h quinone oxidoreductase 1 nqo1

Kidney tissues were prepared using a lysis buffer (20 mM Tris-HCl (pH 7.4), 1% Nonidet P-40 (NP-40), 5 mM ethylenediaminetetraacetic acid (EDTA), 2 mM Na₃VO₄, 100 mM NaF, 10 mM Na₄P₂O₇, 100 μM phenylmethylsulfonyl fluoride (PMSF), 7 μg/mL aprotinin, and 7 μg/mL leupeptin). Proteins were resolved by sodium dodecyl sulfate- (SDS-) polyacrylamide gel electrophoresis and then transferred to polyvinylidene difluoride (PVDF) membrane. After blocking, the membrane was incubated with the following primary antibodies: anti-Bax (Cell Signaling Technology, Danvers, MA, USA), anti-caspase-3 (Cell Signaling Technology), anti-poly(ADP-ribose) polymerase-1 (PARP-1; Cell Signaling Technology), anti-heme oxygenase-1 (HO-1; Santa Cruz Biotechnology), anti-NAD(P)H:quinone oxidoreductase 1 (NQO1; Santa Cruz Biotechnology), and anti-β-actin (Sigma-Aldrich). The membrane was washed and incubated with a horseradish peroxidase-conjugated secondary antibody, and signals were detected using an enhanced chemiluminescence (ECL) Western blotting detection system (ImageQuant LAS4000; GE Healthcare Life Sciences, Pittsburgh, PA, USA). Relative protein expression was quantified using NIH ImageJ software.

Samples were prepared as described previously [95 (link)] and analyzed with 4–12% and 12% precast SDS-PAGE gels (no. NP0321, NP0322 and NP0342, Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instructions. Levels of scanned films (no. 28906837, GE Healthcare, Buckinghamshire, UK) were adjusted in Adobe Photoshop CS4 Extended in accordance with the guidelines for the proper handling of digital image data given in [96 (link)].
The following primary antibodies were used: anti-extracellular signal-regulated kinase 1/2 (ERK1/2) (no. 9102, Cell Signaling, Danvers, MA, USA; a kind gift from Leonard Girnita and Claire Worrall, Karolinska Institutet, Stockholm, Sweden), anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH; no. ab8245, Abcam, Cambridge, UK), anti-NAD(P)H:quinone oxidoreductase 1 (NQO1; no. sc-32793, Santa Cruz, Heidelberg, Germany), anti-Nrf2; no. ab62352, Abcam), anti-p21 (118) [97 (link)], anti-p53 (DO-7) [98 (link)], anti-phospho-ERK1/2 (no. 9101, Cell Signaling; a kind gift from Leonard Girnita and Claire Worrall) and anti-phospho-histone H2AX (Ser 139) (γ-H2AX; no. 05-636, Millipore, Molsheim Cedex, France). Horseradish peroxidase (HRP)-conjugated secondary antibodies were rabbit anti-mouse (no. P0161, Dako, Glostrup, Denmark) and swine anti-rabbit (no. P0211, Dako).