Anti heme oxygenase 1 ho 1 antibody

Dulbecco’s modified Eagle’s medium (DMEM) and fetal bovine serum (FBS) were obtained from Life Technologies (Tokyo, Japan). EPA, DHA, and 4-HHE were purchased from Cayman (Ann Arbor, MI, USA). Anti-β-actin antibody (A5316) and N-acetyl-l-cysteine (NAC) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Fatty acid-free bovine serum albumin (BSA) was purchased from Nacalai Tesque (Kyoto, Japan). Anti-epoxide hydrolase 2 (sEH) antibody was purchased from Gene Tex Inc. (Irvine, CA, USA). Anti-p38 kinase (p38) (#9212) and anti-phospho-p38 (#9211) antibodies were purchased from Cell Signaling (Danvers, MA, USA). Anti-PECAM-1 (M-20) antibody and anti-heme oxygenase 1 (HO-1) antibody were purchased from Santa Cruz (Dallas, TX, USA). Anti-cytochrome P450 2J2 (CYP2J2) antibody was purchased from Origene (Rockville, MD, USA). Horseradish peroxidase (HRP)-conjugated anti-mouse and anti-rabbit antibodies were purchased from Amersham Biosciences Corp. (Piscataway, NJ, USA). Small interfering RNA (siRNA) was purchased from Life Technologies (Tokyo, Japan). SB203580 was purchased from Calbiochem (Cambridge, UK). All other reagents and chemicals were from standard suppliers.

Protein lysates were prepared in EBC lysis buffer containing 1% protease inhibitor (Sigma-Aldrich Co., St. Louis, MO, USA). Equal amounts of total protein were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and then electrotransferred onto nitrocellulose membranes (Bio-Rad Laboratories Inc., Hercules, CA, USA). The blots were incubated with blocking reagent (TBS-T with 4% skim milk) at room temperature for 1 h and then hybridized with primary antibodies overnight at 4 °C. Signals were illuminated using Enhanced Chemiluminescence reagent (Bio-Rad Laboratories Inc., Hercules, CA, USA) and recorded on an Image-Quant LAS-4000 imaging system (GE Healthcare, Chicago, IL, USA). The antibodies used in this study included anti-SESN2 antibody (GeneTex Inc., Alton Pkwy Irvine, CA, USA; dilution = 1:1000), anti-heme oxygenase 1 (HO-1) antibody (Santa Cruz Biotechnology Inc., Dallas, TX, USA; dilution = 1:100), and anti-GAPDH (Thermo Fisher Scientific, Waltham, MA, USA; dilution = 1:5000).