Anti phospho nrf2

Cells were lysed in Laemelli SDS reducing buffer [50 mM Tris-HCl (pH 6.8), 2% SDS, and 10% glycerol], boiled, and separated by SDS/PAGE. The following antibodies were used: anti- HO1, -PRX1, -NQO1 (1∶1000; AbCam), anti- Nrf2 (1∶500 Santa Cruz; 1∶1000 Cell Signaling), anti-phospho-Nrf2 (1∶5000; AbCam), and anti–β-actin (1∶20,000; Sigma).

AA was obtained from Calbiochem (San Diego, CA, USA). Antibodies against procaspase-3, cleaved caspase-3, poly (ADP-ribose) polymerase (PARP), Bcl-2, lamin A/C, ERK1/2, phospho-ERK1/2, and NAD(P)H:quinone oxidoreductase 1 (NQO1) along with horseradish peroxidase-conjugated goat anti-mouse antibodies were provided by Cell Signaling Technology (Beverly, MA, USA). Anti-Nrf2 and anti-cleaved PARP antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). The anti-phospho-Nrf2, anti-glutamate-cysteine ligase catalytic subunit (GCLC), and anti-4-hydroxynonenal (4-HNE) polyclonal antibodies were purchased from Abcam (Cambridge, MA, USA). The Fugene® HD and luciferase assay kit were obtained from Promega (Madison, WI, USA). Anti-nitrotyrosine (NT) polyclonal antibody was purchased from Millipore Corporation (Bedford, MA, USA). The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT), rhodamine 123 (Rh123), 2′,7′-dichlorofluorescein diacetate (DCFH-DA), silymarin (SIL), anti-β-actin antibody, and other reagents were supplied by Sigma-Aldrich (St. Louis, MO, USA).