Anti phospho egfr tyr1068 antibody

Epidermal growth factor (EGF), p-nitrophenyl phosphate (pNPP), and dimethyl sulfoxide (DMSO) were obtained from Sigma-Aldrich (St. Louis, MO, USA). 1,2-NQ (95% purity determined by HPLC), 1,4-NQ (100% purity determined by GC), and 1,4-BQ (99.1% purity determined by iodometric titration) were purchased from Tokyo Chemical Industry (Tokyo, Japan).
PD153035 and anti-PTP1B antibody (# PH01) were acquired from Calbiochem (San Diego, CA, USA). Biotin-(PEAC) 5 -maleimide (BPM) and 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) were obtained from Dojindo Laboratories (Kumamoto, Japan). The anti-EGFR antibody (#2232), anti-phospho EGFR Tyr1068 antibody (#2234), anti-p44/42 MAP kinase antibody [anti-extracellular signal-regulated kinase 1/2 (ERK1/2), #9102], anti-phospho ERK1/2 antibody (#9101), horseradish peroxidase (HRP)-linked anti-rabbit IgG (#7074), and anti-mouse IgG (#7076) secondary antibody were procured from Cell Signaling Technology (Beverly, MA, USA). Fetal bovine serum, GlutaMAX-I, tris(2-carboxyethyl) phosphine hydrochloride (TCEP) and Ni-IDA ProBond were from Corning (Woodland, CA, USA), Gibco (Grand Island, NY, USA), Hampton Research (Aliso Viejo, CA, USA) and Invitrogen (Carlsbad, CA, USA), respectively. The anti-1,4-NQ antibody was prepared as previously reported (Hirose et al., 2012) . All other reagents used were of the highest purity available.

Formalin-fixed paraffin-embedded tissues were sectioned and stained with hematoxylin and eosin. Immunohistochemistry was performed on the sections using anti-phospho-EGFR (Tyr1068) antibody (Cell Signaling Technology, Danvers, MA), anti-phospho-HER2 (Tyr1221/1222) antibody (Cell Signaling Technology), anti-phospho-HER3 (Tyr1289) antibody (Cell Signaling Technology), and anti-phospho-HER4 (Tyr1162) antibody (Abcam, Cambridge, United Kingdom) following the manufacturer's recommendations.