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: Renal Failure

Article Title: LCZ696, an angiotensin receptor-neprilysin inhibitor, ameliorates epithelial-mesenchymal transition of peritoneal mesothelial cells and M2 macrophage polarization

doi: 10.1080/0886022x.2024.2392849

Figure Lengend Snippet: Figure 4. LCZ696 Inhibits TGF-β1-induced EMT through the inactivation of PDGFRβ and EGFR pathway. (A) Western blot was conducted to evaluate the protein level of p-PDGFRβ, PDGFRβ and GAPDH in HPMCs cell lysates. (B) Scatter plot showing the densitometry analysis of p-PDGFRβ normalized by PDGFRβ. (C) Scatter plot showing the densitometry analysis of PDGFRβ normalized by GAPDH. (D) Western blot was conducted to evaluate the protein level of p-EGFR, EGFR and GAPDH in HPMCs cell lysates. (E) Scatter plot showing the densitometry analysis of p-EGFR normalized by EGFR. (F) Scatter plot showing the densitometry analysis of EGFR normalized by GAPDH. Data are expressed as mean ± SEM. **P < 0.01; ***P < 0.001; ****P < 0.0001. N.S., statis tically not significant, with the comparisons labeled.

Article Snippet: Antibodies and reagents antibodies to p-Smad3 (#9520), Smad3 (#9523), E-cadherin (#3195), Snail (#3879), PdGFrβ (#3169), p-PdGFrβ (#3161), Stat6 (#5397), p-Stat6 (#56554), EGFr (#4267) and p-EGFr (#3777) were purchased from Cell Signaling technology. antibodies to GaPdH (sc-32233), Collagen I (a2) (sc-28654) and tGFβrI (sc-399) were purchased from Santa Cruz biotechnology. antibodies to Slug (ab27568), α-SMa (ab5694), Fibronectin (ab2413) and CtGF (ab6992) were purchased from abcam (Cambridge, Ma). antibody to Collagen III (a0817) was purchased from abclonal (Wuhan, China). antibodies to arginase-1 (Gb11285) and Cd163 (Gb11340) were purchased from Servicebio (Wuhan, China). anti-mouse secondary antibody (a0216), and anti-rabbit secondary antibody (a0208) were purchased from beyotime Institute of biotechnology (Shanghai, China). tGF-β1 ELISa Kit (rK00057) and IL-4 ELISa Kit (rK00003) were purchased from abclonal (Wuhan, China). tGF-β1 protein and IL-4 protein were purchased from r&d Systems (Minneapolis, MN, united States).

Techniques: Western Blot, Labeling

Journal: Genes to cells : devoted to molecular & cellular mechanisms

Article Title: Endothelial cell-specific molecule 2 (ECSM2) modulates actin remodeling and epidermal growth factor receptor signaling.

doi: 10.1111/j.1365-2443.2008.01267.x

Figure Lengend Snippet: Figure 6 ECSM2-GFP retards the EGF-induced cell migration. (A) Co-expression of EGFR and ECSM2-GFP in HEK293 cells, verified by immunoblotting with anti-EGFR and anti-GFP antibodies (left panel), and by visualization of green fluorescence (right panel). EGFR/ECSM2-GFP-coexpressing cells exhibited substantial filopodia-like structures (arrows). (B) Wound closure assays were performed in HEK293 cells expressing EGFR alone or co-expressing EGFR/ECSM2-GFP. Images were captured at 0–48 h after wounding in the serum-free media with (+) or without (−) EGF (1 nm). Notably, ECSM2-GFP co-expression markedly inhibited the EGF-induced cell migration 24 h after wounding (e vs. f). (C) The average width of wound under each condition was plotted at various time points as indicated, in which the wound width for EGFR-expressing cells at 0 h (as shown in a) was set as 100%. Data are mean ± SE (n = 10). **P < 0.01.

Article Snippet: Anti-phospho-EGFR antibodies (Tyr-845, Tyr-1045, and Tyr-1068) were from Cell Signaling (Beverly, MA).

Techniques: Migration, Expressing, Western Blot, Fluorescence

Journal: Genes to cells : devoted to molecular & cellular mechanisms

Article Title: Endothelial cell-specific molecule 2 (ECSM2) modulates actin remodeling and epidermal growth factor receptor signaling.

doi: 10.1111/j.1365-2443.2008.01267.x

Figure Lengend Snippet: Figure 7 ECSM2 suppresses EGFR signaling. EGFR-expressing or EGFR/ ECSM2-GFP-coexpressing HEK293 cells in the absence (lanes 1 and 2) or presence (lanes 3 and 4) of EGF (1 nm) were sub- jected to assessment of tyrosine phosphor- ylation of EGFR (A), activation of Shc (B), and ERK (C) by immunoblotting with anti-phospho-EGFR (pY-845, pY-1045, and pY-1068), anti-phospho-Shc (pShc), and anti-phospho-ERK (pERK) antibodies, respectively. Immunoblotting with anti- total EGFR, Shc, and ERK antibodies, as indicated, verified the equal loadings in each panel. (D and E) Data of pShc and pERK from three independent experiments were subjected to densitometric analysis, respectively. The pShc or pERK level in EGFR-expressing cells in the absence of EGF (lane 1 in B or C) was considered as 100%. Data are mean ± SE *P < 0.05; **P < 0.01. Experiment shown in A is representative of three such experiments.

Article Snippet: Anti-phospho-EGFR antibodies (Tyr-845, Tyr-1045, and Tyr-1068) were from Cell Signaling (Beverly, MA).

Techniques: Expressing, Activation Assay, Western Blot