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: The Journal of biological chemistry

Article Title: Enhanced tumorigenic behavior of glioblastoma cells expressing a truncated epidermal growth factor receptor is mediated through the Ras-Shc-Grb2 pathway.

doi: 10.1074/jbc.271.41.25639

Figure Lengend Snippet: FIG. 2. Interaction of phosphoproteins with Shc in U87MG cells expressing mutant EGFRs. A, all mutants analyzed contained a truncation of 801 base pairs in the EGFR extracellular domain. The D (DEGFR) mutant contained intact phosphorylation sites; DK contained a lysine to methionine point mutation at the ATP binding site (K721); DY1, DY2, DY3, DY4, and DY5 contained tyrosine to phenylalanine substitutions at known phosphorylation sites as indicated, where X denotes a mutated site. B, lysates prepared from EGF stimulated (1) and unstimulated (2) U87MG cells expressing truncated EGFR mu- tants were analyzed for the presence of the truncated receptors (a) and for the presence of tyrosine-phosphorylated proteins (b). Shc immuno- precipitates prepared from these cells were probed with an anti-phos- photyrosine antibody to detect the presence of phosphorylated mutant EGF receptors (c), and phosphorylated Shc (d). Shc precipitates were also analyzed for the presence of Grb2 associated with Shc (e)

Article Snippet: The monoclonal antibody used for specific precipitation of the truncated EGF receptor was raised against cells expressing the truncated EGFR and will be described elsewhere,2 and the antibody for precipitation of endogenous receptor was EGFR1 (Santa Cruz, sc-101).

Techniques: Expressing, Mutagenesis, Phospho-proteomics, Binding Assay

Journal: The Journal of biological chemistry

Article Title: Enhanced tumorigenic behavior of glioblastoma cells expressing a truncated epidermal growth factor receptor is mediated through the Ras-Shc-Grb2 pathway.

doi: 10.1074/jbc.271.41.25639

Figure Lengend Snippet: FIG. 3. Interaction of Shc and Grb2 with truncated EGFR mu- tants. Lysates were prepared from U87MG cells expressing truncated receptor mutants, and U87MG cells expressing wild-type EGFR (U87MG.wtEGFR), which had been pretreated with EGF (1) or were untreated (2). Total lysate was analyzed to demonstrate the difference in phosphorylation state of the receptor mutants (b). Truncated EGFR was precipitated from equal amounts of total protein using an antibody specific for the mutant receptor, except in the case of the U87MG.wtEGFR cells where an antibody recognizing the full-length receptor was used. Immunoprecipitates were analyzed for EGFR (a) Shc (c) and Grb2 (d) by Western blotting.

Article Snippet: The monoclonal antibody used for specific precipitation of the truncated EGF receptor was raised against cells expressing the truncated EGFR and will be described elsewhere,2 and the antibody for precipitation of endogenous receptor was EGFR1 (Santa Cruz, sc-101).

Techniques: Expressing, Phospho-proteomics, Mutagenesis, Western Blot