Phosphorylated egfr

The western blotting assay was applied to study the proteins related to the EGFR-Ras-Raf signaling pathway. Proteins were extracted and subjected to 10% SDS-PAGE, then transferred to PVDF membranes. The membranes were blocked with 5% skim milk in Tris-buffered saline with Tween™20 (Sigma-Aldrich) for 1 h at room temperature. The membranes were reacted with primary antibodies overnight at 4 °C. The primary antibodies were as follows: phosphorylated EGFR (1:500); Ras (1:500); phosphorylated Raf (1:500); phosphorylated Erk1/2 (1:500, all from Cell Signaling Technology, Danvers, MA, USA); and β-actin (Santa Cruz Biotechnology, 1:1,000). β-actin protein was used as the control.

Low passage log-phase cells were plated into 6-well plates and incubated overnight. Cells were incubated with 1ml 100nM antibodies or DVD-Ig proteins for 24 hours in the presence of 3nM HRG. Cells were then harvested and lysed with RIPA buffer (Sigma-Aldrich, MO) supplemented with protease and phosphatase inhibitor cocktail tablet according to the manufacturer's instructions (Roche Diagnostics, IN). Cell lysate proteins were resolved by SDS-PAGE and immunoblots were probed with antibodies against phosphorylated EGFR(Tyr¹⁰⁶⁸; Cat#3777), ErbB2(Tyr^1221/1222; Cat#2243), and ErbB3(Tyr¹¹⁹⁷; Cat#4561) (Cell Signaling, MA), EGFR (NeoMarker,Thermo Fisher, CA), ErbB2 and ErbB3 (Cell Signaling, MA), phosphorylated extracellular signal-regulated kinase (pERK) 1/2 (Thr²⁰²/Tyr²⁰⁴; Cat#4370) (Cell Signaling, MA), ERK1/2, AKT (Santa Cruz, CA), ErbB2 (Cell Signaling, CA), phosphorylated AKT (pAKT; Ser⁴⁷³; Cat#4060) (Cell Signaling, MA), followed by incubation with IRDye 700 conjugated goat anti-mouse and IRDye 800 conjugated goat anti-rabbit (LI-COR, NE). Total protein was normalized with anti-PCNA (Santa Cruz, CA) followed by IRDye680 CW conjugated goat anti-mouse (LI-COR, NE). Blots were visualized using an Odyssey Imaging system (LI-COR, NE).

Immunoblotting analysis was performed using whole cell lysates prepared in RIPA buffer (50 mM Tris–HCl, pH 7.4, 150 mM NaCl, 2 mM EDTA, 1% NP-40, 10% glycerol) plus a protease inhibitor cocktail (Sigma, St Louis, MO). Nuclear protein was extracted using NE-PER Nuclear and Cytoplasmic Extraction Reagents from Pierce Biotechnology (Rockford, IL). Antibodies anti-p65 (Cat# sc-8008), p50 (Cat# sc-7178), FOXC1 (Cat# sc-21,394), LaminA/C (Cat# sc-7292), β-actin (Cat# sc-8432), GAPDH (Cat# sc-47,724) are from Santa Cruz Biotechnology (Santa Cruz, CA). Antibodies anti-FOXC1 (Cat# 8758), EGFR (Cat# 4267), phosphorylated-EGFR (Cat# 3777), phospho-NF-κB p65 (Ser536) (Cat# 3033) are from Cell Signaling Technology.

Lapatinib (GSK572016), gefitinib (ZD1839), and linsitinib (OSI-906) were purchased from TopScience (Shanghai, China), dissolved in dimethyl sulfoxide (DMSO), and stored at a concentration of 10 mmol/L. ((3-(4, 5-Dimethyl-thiazol-2-yl)-2, 5-diphenyltetrazolium bromide) (MTT) was obtained from Sigma-Aldrich Chemical Inc. (St. Louis, MO, USA). The primary antibodies, including phosphorylated -EGFR, -HER2, -IGF-1R, -AKT (Ser473), -p44/42MPAK (ERK), and -β-actin, were provided by Cell Signaling Technology (Danvers, MA, USA). The Annexin V-FITC Apoptosis Detection Kit and cell cycle and apoptosis analysis kit were supplied by Beyotime Biotechnology (Nantong, Jiangsu, China). The Matrigel invasion chamber 24-well plate 8.0 micron was purchased from Corning (Corning, NY, USA).

The whole-cell protein lysates or animal tumor tissue specimens were prepared as previously described [47 (link)] and analyzed by western blot with the following primary antibodies: phosphorylated STAT3 (p-STAT3, Tyr705), phosphorylated AKT (p-AKT, Ser473), phosphorylated MAPK (p-MAPK, Thr202/Tyr204), phosphorylated HER2 (p-HER2, Tyr1221/1222), phosphorylated EGFR (p-EGFR, Tyr1068), phosphorylated HER3 (p-HER3, Tyr1289), phosphorylated IGF-IR (p-IGF-IR, Tyr1316), STAT3, AKT, MAPK, EGFR, HER2, HER3, IGF-IR, PTEN, caspase 3 and 7 from Cell Signaling Technology (Denvers, MA, USA); FN, IL-6, IL-10, LIF, EGF, MUC1 (clone EP1024Y) and MUC4 (clone 8G-7) from Abcam (Cambridge, MA, USA).