Monoclonal phospho tyrosine antibody p tyr 100

NR6WT fibroblasts, originally constructed by stably expressing human EGFR in murine 3T3 fibroblasts that were selected to lack endogenous EGFR [14 (link), 15 (link)] were grown in alpha-MEM media supplemented with 7.5% fetal bovine serum, 1x non-essential amino acids, 1x sodium pyruvate, 1x L-glutamine and 1x pen/strep (100 units/ml penicillin; 100 μg/ml streptomycin) antibiotics. Melanoma cell line WM1158 and WM983b cells were cultured in DMEM (1 gL^-1 glucose) : L-15 3:1 medium containing 10% fetal bovine serum and 1x pen/strip antibiotics. Plasmid transfection reagent xFect, Plasmid isolation kit and PCR extraction kit were purchased from Clontech/Takara Life Technologies (Grand Island, NY). Monoclonal GFP antibody conjugated to agarose beads (GFP-Trap A) was purchased from ChromoTek GmbH Planegg-Martinsried, Germany). Monoclonal EGFR antibody conjugated agarose (EGFR antibody (A-10) AC, normal mouse IgG-AC and monoclonal GFP were purchased from Santa Cruz (Dallas, TA). Monoclonal phospho-tyrosine antibody (p-Tyr-100) and polyclonal antibodies against phospho-EGFR (Y1173) and phospho-p38, phospho-ERK (Thr202/Tyr204) were purchased from Cell Signaling Technology (Beverly, MA). AccuPrime™ Pfx SuperMix was purchased from Invitrogen/Thermo Fisher Scientific (Carlsbad, CA). Restriction enzymes were purchased from New England Biolabs (Ipswitch, MA).