Journal: Journal of Carcinogenesis

Article Title: Sulindac metabolites inhibit epidermal growth factor receptor activation and expression

doi: 10.1186/1477-3163-4-16

Figure Lengend Snippet: EGF induces EGFR and ERK1/2 phosphorylation . HT29 human colon cancer cells were grown to 80% confluence in medium containing 10% FBS then serum deprived for 48 h before treatment with vehicle (water), 10, or 100 ng/ml EGF. Cells were harvested 10 min after EGF treatment and lysates prepared for (A) Immunoblotting with antibodies raised against pEGFR (pY1068), total EGFR, pERK1/2, and total ERK1/2; α-tubulin immunoblots of the same lysates served as loading controls. The graphs show the densitometry results of the pEGFR bands (B) and pERK1/2 bands (C) normalized for the loading controls. Data represent mean of triplicate samples ± SD; statistical significance is denoted by ***p < 0.001 versus vehicle. Results shown in the figure are representative of 2 separate experiments each with triplicate samples.

Article Snippet: Primary antibodies raised against phosphorylated ERK1/2 (Thr202/Tyr204), total ERK1/2, and total EGFR were purchased from Santa Cruz Biotechnology (Santa Cruz, CA); primary antibody against phosphorylated EGFR(Tyr1068), horseradish peroxidase-conjugated anti-mouse and anti-rabbit secondary antibodies, and EGF were purchased from Biosource (Camarillo, CA).

Techniques: Western Blot

Journal: Journal of Carcinogenesis

Article Title: Sulindac metabolites inhibit epidermal growth factor receptor activation and expression

doi: 10.1186/1477-3163-4-16

Figure Lengend Snippet: Dose response of sulindac sulfide inhibition of EGFR . HT29 cells were grown to 80% confluence in medium containing 10% FBS then serum deprived for 24 h before addition of drug. Cells were then treated with vehicle (0.1% DMSO), 40, 80, 120, or 160 μM sulindac sulfide, drug doses previously shown to induce apoptotic cell death in these cells. Twenty four hours after drug treatment, vehicle or 10 ng/ml EGF was added and cells were harvested 10 min later. (A) Immunoblots were performed on cell lysates with antibodies raised against pEGFR (pY1068), total EGFR, pERK1/2, total ERK1/2, and caspase-3; total ERK1/2 immunoblots served as loading controls. The graphs show the densitometry results of the pEGFR bands (B) and total EGFR bands (C) . Results shown in figure are representative of 3 separate experiments.

Article Snippet: Primary antibodies raised against phosphorylated ERK1/2 (Thr202/Tyr204), total ERK1/2, and total EGFR were purchased from Santa Cruz Biotechnology (Santa Cruz, CA); primary antibody against phosphorylated EGFR(Tyr1068), horseradish peroxidase-conjugated anti-mouse and anti-rabbit secondary antibodies, and EGF were purchased from Biosource (Camarillo, CA).

Techniques: Inhibition, Western Blot

Journal: Journal of Carcinogenesis

Article Title: Sulindac metabolites inhibit epidermal growth factor receptor activation and expression

doi: 10.1186/1477-3163-4-16

Figure Lengend Snippet: Dose response of sulindac sulfone inhibition of EGFR . HT29 cells were grown to 80% confluence in medium containing 10% FBS then serum deprived for 24 h before addition of drug. Cells were then treated with vehicle (0.2% DMSO), 200, 400, 600, or 800 μM sulindac sulfone, drug doses previously shown to induce apoptotic cell death in these cells. Twenty four hours after drug treatment, vehicle or 10 ng/ml EGF was added and cells were harvested 10 min later. (A) Immunoblots were performed on cell lysates with antibodies raised against pEGFR (pY1068), total EGFR, pERK1/2, total ERK1/2, and caspase-3; total ERK1/2 immunoblots served as loading controls. The graphs show the densitometry results of the pEGFR bands (B) and total EGFR bands (C) . Results shown in figure are representative of 3 separate experiments.

Article Snippet: Primary antibodies raised against phosphorylated ERK1/2 (Thr202/Tyr204), total ERK1/2, and total EGFR were purchased from Santa Cruz Biotechnology (Santa Cruz, CA); primary antibody against phosphorylated EGFR(Tyr1068), horseradish peroxidase-conjugated anti-mouse and anti-rabbit secondary antibodies, and EGF were purchased from Biosource (Camarillo, CA).

Techniques: Inhibition, Western Blot

Journal: Journal of Carcinogenesis

Article Title: Sulindac metabolites inhibit epidermal growth factor receptor activation and expression

doi: 10.1186/1477-3163-4-16

Figure Lengend Snippet: Dose response and time course of sulindac sulfide inhibition of EGFR . HT29 cells were grown to confluence in medium containing 10% FBS and treated with vehicle (0.1% DMSO), 160, or 180 μM sulindac sulfide for 1 h, 12 h, and 24 h. Cells were then harvested and immunoblots were performed on cell lysates with antibodies raised against pEGFR (pY1068), total EGFR, pERK1/2, total ERK1/2, and cleaved caspase-3; α-tubulin immunoblots of the same lysates served as loading controls. (A) 1 h, 12 h, and 24 h immunoblot results. The graphs show the densitometry results of the pEGFR bands (B) and total EGFR bands (C) . Data represent mean of triplicate samples ± SD; statistical significance is denoted by **p < 0.01 and ***p < 0.001 versus respective time point vehicle. Results shown in figure are representative of 2 separate experiments each with triplicate samples.

Article Snippet: Primary antibodies raised against phosphorylated ERK1/2 (Thr202/Tyr204), total ERK1/2, and total EGFR were purchased from Santa Cruz Biotechnology (Santa Cruz, CA); primary antibody against phosphorylated EGFR(Tyr1068), horseradish peroxidase-conjugated anti-mouse and anti-rabbit secondary antibodies, and EGF were purchased from Biosource (Camarillo, CA).

Techniques: Inhibition, Western Blot

Journal: Journal of Carcinogenesis

Article Title: Sulindac metabolites inhibit epidermal growth factor receptor activation and expression

doi: 10.1186/1477-3163-4-16

Figure Lengend Snippet: Dose response and time course of sulindac sulfone inhibition of EGFR . HT29 cells were grown to confluence in medium containing 10% FBS followed by treatment with vehicle (0.2% DMSO), 400, or 600 μM sulindac sulfone for 1 h, 12 h, and 24 h. Cells were then harvested and immunoblots were performed on cell lysates with antibodies raised against pEGFR (pY1068), total EGFR, pERK1/2, total ERK1/2, and cleaved caspase-3; α-tubulin immunoblots of the same lysates served as loading controls. (A) 1 h, 12 h, and 24 h Western blot results. The graphs show the densitometry results of the pEGFR bands (B) and total EGFR bands (C) . Data represent mean of triplicate samples ± SD; statistical significance is denoted by **p < 0.01 and ***p < 0.001 versus respective time point vehicle. Results shown in figure are representative of 2 separate experiments each with triplicate samples.

Article Snippet: Primary antibodies raised against phosphorylated ERK1/2 (Thr202/Tyr204), total ERK1/2, and total EGFR were purchased from Santa Cruz Biotechnology (Santa Cruz, CA); primary antibody against phosphorylated EGFR(Tyr1068), horseradish peroxidase-conjugated anti-mouse and anti-rabbit secondary antibodies, and EGF were purchased from Biosource (Camarillo, CA).

Techniques: Inhibition, Western Blot

Journal: Journal of Carcinogenesis

Article Title: Sulindac metabolites inhibit epidermal growth factor receptor activation and expression

doi: 10.1186/1477-3163-4-16

Figure Lengend Snippet: Effect of the caspase inhibitor, ZVAD, on apoptosis and inhibition of EGFR . HT29 colon cancer cells were grown to confluence in medium containing 10% FBS followed by pretreatment with or without 25 μM zvad for 1 h. Cells were then treated with vehicle (0.2% DMSO) or 600 μM sulfone for 48 h. Cells were harvested and immunoblots were performed on cell lysates with antibodies raised against pEGFR (pY1068), total EGFR, and cleaved caspase 3; α-tubulin immunoblots of the same lysates served as loading controls. The graphs show morphological apoptosis results (A) 48 h Western immunoblot results (B) and densitometry of the pEGFR bands (C) and total EGFR bands (D) . Data represent mean of triplicate samples ± SD; statistical significance is denoted by *p < 0.05, **p < 0.01 and ***p < 0.001. Results shown in figure are representative of 2 separate experiments each with triplicate samples.

Article Snippet: Primary antibodies raised against phosphorylated ERK1/2 (Thr202/Tyr204), total ERK1/2, and total EGFR were purchased from Santa Cruz Biotechnology (Santa Cruz, CA); primary antibody against phosphorylated EGFR(Tyr1068), horseradish peroxidase-conjugated anti-mouse and anti-rabbit secondary antibodies, and EGF were purchased from Biosource (Camarillo, CA).

Techniques: Inhibition, Western Blot

Journal: Journal of the American Society of Nephrology

Article Title: Albumin Activates ERKViaEGF Receptor in Human Renal Epithelial Cells

doi: 10.1681/asn.2004030222

Figure Lengend Snippet: Figure 8. The effect of albumin on phosphorylation of the EGF receptor (EGFR). The phosphorylation of the EGFR in response to albumin (1%) and the ligand, EGF (100 ng/ml), was compared in the cultured human kidney cells. Albumin led to the phosphorylation of the EGFR on tyrosine 845 and tyrosine 1068 (as did EGF), and the phosphorylation of both of these residues was prevented by AG1478 and PD1587780 in the albumin-treated cells. *P0.05 compared with control cells. The data are representative of three separate experiments.

Article Snippet: Phospho-EGF receptor (Tyr 1068 and Tyr 845) antibody was purchased from Cell Signaling Technology (Beverley, MA).

Techniques: Phospho-proteomics, Cell Culture, Control

Journal: Cell reports

Article Title: Glioblastoma Cell Resistance to EGFR and MET Inhibition Can Be Overcome via Blockade of FGFR-SPRY2 Bypass Signaling

doi: 10.1016/j.celrep.2020.02.014

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Phospho-EGF Receptor (Tyr1068) (D7A5) XP Rabbit mAb , Cell Signaling Technology , RRID:AB_2096270.

Techniques: Virus, Subcloning, Recombinant, Chromatin Immunoprecipitation, Ab Array, Enzyme-linked Immunosorbent Assay, Proliferation Assay, Bicinchoninic Acid Protein Assay, Stable Transfection, Expressing, shRNA, Sequencing, Software

Journal: Frontiers in Cell and Developmental Biology

Article Title: BML-265 and Tyrphostin AG1478 Disperse the Golgi Apparatus and Abolish Protein Transport in Human Cells

doi: 10.3389/fcell.2019.00232

Figure Lengend Snippet: BML-265 and Tyrphostin AG1478 inhibit the transport of secretory proteins. HeLa cells transiently expressing Str-KDEL_TNF-SBP-EGFP (A) , SBP-EGFP-GPI (B) or SBP-EGFP-EGFR (C) were pre-treated with the indicated molecules at 10 μM for 1 h. Trafficking of the reporters was then induced by incubation with biotin for 1 h. The presence of the GFP-tagged reporters (green, upper panel) at the plasma membrane was then detected using an anti-GFP antibody on non-permeabilized cells (red, upper panel). The Golgi apparatus was visualized using immunostaining against GM130 (bottom panel). Scale bar: 10 μm.

Article Snippet: Antibodies to detect phosphorylated EGFR and total EGFR were purchased from Cell Signaling Technology (PhosphoPlus EGFR (Tyr1068) Antibody Duet, catalog number 11862S, dilution 1:1000).

Techniques: Expressing, Incubation, Immunostaining