Journal: Nature Communications

Article Title: Quantification of ligand and mutation-induced bias in EGFR phosphorylation in direct response to ligand binding

doi: 10.1038/s41467-023-42926-8

Figure Lengend Snippet: A Raw dose response curves for Y1068 and Y1173 phosphorylation per EGFR molecule, for the ligands EGF, TGFα, and epiregulin. Each point represents the ratio of either anti-pY1068 or anti-pY1173 fluorescence and EGFR-mTurq fluorescence for one individual vesicle. Each curve contains ~1000 to ~3000 data points (single vesicles). B Bias plots. Shown are means (symbols) and standard errors (often smaller than symbols). The epiregulin points diverge from the EGF and TGFα points. In total, data are from 11,570 single vesicles over 25 independent experiments. C Bias coefficients and standard errors. Epiregulin is biased toward Y1173 phosphorylation as compared to EGF and TGFα. Ordinary one-way ANOVA, followed by Tukey’s test, was used to determine statistical significance. The p values are adjusted for multiple comparisons.

Article Snippet: Y1173 phosphorylation was detected using 50 nM AlexaF488-labeled anti-pY1173 EGFR antibody (NBP1-44893AF488, Novus Biologicals) .

Techniques: Phospho-proteomics, Fluorescence

Journal: Nature Communications

Article Title: Quantification of ligand and mutation-induced bias in EGFR phosphorylation in direct response to ligand binding

doi: 10.1038/s41467-023-42926-8

Figure Lengend Snippet: A Single vesicle dose response curves for Y1068 and Y1173 phosphorylation per EGFR molecule, for EGF, TGFα, and epiregulin. Each point represents the ratio of either anti-pY1068 or anti-pY1173 fluorescence and EGFR-mTurq fluorescence for one individual vesicle. Each curve contains ~1000 to ~1800 data points. B Bias plots. Shown are means (symbols) and standard errors (often smaller than symbols). In total, data are from 8009 vesicles in 23 independent experiments. C Bias coefficients and their standard errors. Ordinary one-way ANOVA, followed by Tukey’s test, was used to determine statistical significance. The p values are adjusted for multiple comparisons.

Article Snippet: Y1173 phosphorylation was detected using 50 nM AlexaF488-labeled anti-pY1173 EGFR antibody (NBP1-44893AF488, Novus Biologicals) .

Techniques: Phospho-proteomics, Fluorescence

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: Drug Design, Development and Therapy

Article Title: Blockade of the P2Y2 Receptor Attenuates Alcoholic Liver Inflammation by Targeting the EGFR-ERK1/2 Signaling Pathway

doi: 10.2147/DDDT.S346376

Figure Lengend Snippet: Hepatoprotective effect of suramin was associated with inhibiting EGFR-ERK1/2 signaling pathway. ( A – D ) The protein expression of P2Y2R, TNF-α and IL-1β in the liver. ( E and F ) The protein expression levels of EGFR, p-EGFR, ERK1/2 and p-ERK1/2 in the liver. *** P < 0.001 compared to control group. ## P < 0.01, ### P < 0.001 compared to Alcohol group.

Article Snippet: Antibodies against total ERK, phospho-ERK1/2 (p-ERK1/2), total EGFR and phospho-EGFR (p-EGFR) were obtained from Elabscience (Wuhan, China).

Techniques: Expressing, Control

Journal: Drug Design, Development and Therapy

Article Title: Blockade of the P2Y2 Receptor Attenuates Alcoholic Liver Inflammation by Targeting the EGFR-ERK1/2 Signaling Pathway

doi: 10.2147/DDDT.S346376

Figure Lengend Snippet: ERK1/2 inhibition mitigated alcohol-induced AML-12 cell inflammation. ( A and B ) The effect of UTP (1.89 μM) and suramin (200 μM) on alcohol-induced phosphorylation of ERK1/2 in AML-12 cell. ( E and F ) The effect of the EGFR antagonist AG1478 (3 nmol/L) on alcohol-induced phosphorylation of ERK1/2 in AML-12 cell. ( G and H ) The effect of the ERK1/2 antagonist U0126 (65 nmol/L) on alcohol-induced phosphorylation of ERK1/2 in AML-12 cell. ( I – K ) The effect of the ERK1/2 antagonist U0126 (65 nmol/L) on alcohol-induced protein and mRNA expression of TNF-α and IL-1β in AML-12 cell. ** P < 0.01, *** P < 0.001 compared to the control group. # P < 0.05, ## P < 0.01 compared to the alcohol group, && P < 0.01 compared to the alcohol plus UTP group. ( C and D ) The effect of P2Y2R silencing and UTP on alcohol-induced phosphorylation of ERK1/2 in AML-12 cell. ** P < 0.01 compared to the control group. ## P < 0.01 compared to the negative control group.

Article Snippet: Antibodies against total ERK, phospho-ERK1/2 (p-ERK1/2), total EGFR and phospho-EGFR (p-EGFR) were obtained from Elabscience (Wuhan, China).

Techniques: Inhibition, Phospho-proteomics, Expressing, Control, Negative Control

Journal: Drug Design, Development and Therapy

Article Title: Blockade of the P2Y2 Receptor Attenuates Alcoholic Liver Inflammation by Targeting the EGFR-ERK1/2 Signaling Pathway

doi: 10.2147/DDDT.S346376

Figure Lengend Snippet: Blockade of the P2Y2 receptor attenuates alcoholic liver inflammation by targeting the EGFR-ERK1/2 signaling pathway.

Article Snippet: Antibodies against total ERK, phospho-ERK1/2 (p-ERK1/2), total EGFR and phospho-EGFR (p-EGFR) were obtained from Elabscience (Wuhan, China).

Techniques: