Mouse monoclonal anti phospho erk1 2

Samples containing 40mg of protein from lysates of harvested PDX were separated by SDS-PAGE on 10% Tris-HCl gels and subsequently transferred to nitrocellulose filters. After blocking (Odyssey Blocking buffer; LI-COR Biosciences (Lincoln, NE)) the blots were incubated with the following primary antibodies: rabbit monoclonal anti-phospho-Akt (1:1000, #4060, Cell Signalling), mouse monoclonal anti- phospho- Erk1/2 (1:1000, #4374, Cell Signalling), mouse monoclonal anti-phosphotyrosine (1:1000, 05-321X, EMD Milipore (Darmstadt, Germany) and rabbit polyclonal anti-Vinculin (1:2, 500, PA5-19842, Thermo Scientific). Subsequently the filters were washed with phosphate-buffered saline (PBS) containing 0.1% Tween and then incubated with Alexa Fluor secondary antibodies (1:5, 000; Invitrogen). Specific proteins were detected using Odyssey IR imaging system (LI-COR Biosciences).

Lipopolysaccharide (LPS) (rough strains) from Escherichia coli F583 (Rd mutant) and fetal bovine serum (FBS) were obtained from Sigma-Aldrich (St. Louis, MO). FBS (lot no. 12J002) was qualified USA origin, sterile, filtered and cell culture tested, and certified to contain endotoxin levels less than 1.0 ng/ml. Dulbecco's modified Eagle's medium (DMEM), penicillin, streptomycin, 0.05% (w/v) trypsin/EDTA, and phosphate-buffered saline (PBS) were obtained from GIBCO (Gaithersburg, MD). Tin Protoporphyrin IX dichloride (TinPPIX) was from Santa Cruz Biotechnology (Santa Cruz, CA). Kinase inhibitors (U0126 and SB202190) were from Cell Signaling (Beverly, MA).
Antibodies used for Western blots include: goat anti-rabbit IgG-horseradish peroxidase, goat anti-mouse IgG-horseradish peroxidase, rabbit polyclonal anti-Nrf2, and anti-HO-1 (Santa Cruz Biotechnology, Santa Cruz, CA); mouse monoclonal anti-β-actin (Sigma-Aldrich, St. Louis, MO); rabbit polyclonal anti-ERK1/2, mouse monoclonal anti-phospho-ERK1/2, rabbit monoclonal anti-p38MAPK, and anti-phospho-p38MAPK (Cell Signaling, Beverly, MA). Quercetin (Sigma-Aldrich, St. Louis, MO) and cyanidin chloride (Indofine Chemical Comp., Hillsborough, NJ) were dissolved in DMSO as a stock solution.

Dulbecco’s modified Eagle’s medium (DMEM), penicillin, streptomycin, 0.05% (w/v) trypsin/EDTA, and phosphate-buffered saline (PBS) were obtained from GIBCO (Gaithersburg, MD). Interferon-γ (IFNγ) was purchased from R & D Systems (Minneapolis, MN). Lipopolysaccharide (LPS) (rough strains) from Escherichia coli F583 (Rd mutant) and methylthiazolyldiphenyl-tetrazolium bromide (MTT) were from Sigma-Aldrich (St. Louis, MO). WST-1 kit for assay of cell viability was obtained from Clontech (Mountain View, CA). Fetal bovine serum was from Atlanta Biologicals (Lawrenceville, GA). Antibodies used for Western blots include: goat anti-rabbit IgG- horseradish peroxidase, goat anti-mouse IgG- horseradish peroxidase and anti-iNOS rabbit polyclonal (Santa Cruz Biotechnology, Santa Cruz, CA); monoclonal anti-β-actin peroxidase (Sigma-Aldrich, St. Louis, MO); STAT1α rabbit polyclonal antibody (Millipore, Billerica, MA), rabbit polyclonal p-STAT1 pSer727 (Pierce Biotechnology, Rockford, IL), rabbit polyclonal anti-ERK1/2, and mouse monoclonal anti-phospho-ERK1/2, (Cell Signaling, Beverly, MA). For ROS detection, CM-H2DCF-DA (DCF) was obtained from Invitrogen, Inc. (Eugene, OR), and dihydroethidium (DHE) from Sigma-Aldrich (St. Louis, MO).