Heme oxygenase ho 1

Cells were lysed with ice-cold PRO-PREP™ protein extract solution (iNtRON, Sungnam, Gyunggi, Korea) and the protein concentration was determined by the BCA assay (Thermo Scientific). Equal amounts of protein were separated by SDS-PAGE and then transferred onto a polyvinylidene difluoride membrane (Millipore, Billerica, MA, USA). The membrane was blocked with 5% skim milk in Tris-HCl (100 mM, pH 7.5), NaCl (150 mM), and 0.2% Tween-20 (TBST) for 1 h at room temperature. The membranes were incubated with TBST containing 5% skim milk and primary antibodies against Nrf2, transcription factor II B (TFIIB), NQO1, GAPDH, and α-tubulin (Santa Cruz Biotechnology, Santa Cruz, CA, USA); heme oxygenase (HO-1, Enzo Life Sciences, Farmingdale, NY, USA); cysteine dioxygenase (CDO) and GCLC (Abcam). After washing with TBST, the blot was incubated with the appropriate horseradish peroxidase (HRP)-conjugated secondary antibodies and visualized using an enhanced chemiluminescent HRP substrate kit (Western Bright, Advansta, Menlo Park, CA, USA).

Formalin fixed slides were analyzed following hematoxylin and eosin (H&E) or Picro-Sirius-Red (PSR) staining. For assessing inflammation and carbonylation, the following primary antibodies were used for IHC: rabbit polyclonal 4-HNE, rabbit polyclonal acrolein (Cell Sciences, Newburyport, MA), rat polyclonal F4/80 (Biorad, Hercules, CA), rat polyclonal B220 (BD Biosciences San Jose, CA), rabbit polyclonal CD3 (DAKO/Agilent Santa Clara, CA), rabbit polyclonal heme oxygenase (HO-1, Enzo Life Sciences, Farmingdale, NY), rabbit polyclonal phospho H2A.X (Cell Signaling, MA), rabbit polyclonal NQO-1 (Sigma, St. Louis, MO), goat polyclonal GSTμ (ABCAM,), rabbit polyclonal GSTπ, (MBL International, Woburn, MA), goat polyclonal myeloperoxidase (MPO) (Millipore, Billerica, MA)[10 (link)], rabbit polyclonal Glutathione Peroxidase 1 (Gpx1) (ABCAM, Cambridge, MA) and rabbit polyclonal Glutamate-Cysteine Ligase Catalytic Subunit (GCLC, Novus Biologicals, Littleton, CO), rabbit polyclonal Cbr1/3 (this study). Histologic images were captured on an Olympus BX51 microscope equipped with a four-megapixel Macrofire digital camera (Optronics; Goleta, CA) using the PictureFrame Application 2.3 (Optronics). All images were cropped and assembled using Photoshop CS2 (Adobe Systems, Inc.; Mountain View, CA).