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: Oncogene

Article Title: Role of AMPK in UVB-induced DNA damage repair and growth control

doi: 10.1038/onc.2012.279

Figure Lengend Snippet: AMPK is not required for inhibiting the ERK pathway by AICAR and metformin. A, immunoblot analysis of p-ACC, ACC, p-ERK, ERK, and GAPDH in mouse skin at 24 h after the final topical treatment with vehicle, AICAR (1 μmol) or metformin (2 μmol) for 23 weeks. B, immunoblot analysis of Cyclin D1, p-ERK, ERK, p-ACC and GAPDH in NHEK cells at 24 h after treatment with vehicle, AICAR (1 mM) or metformin (2 mM). C, immunoblot analysis of AMPK, p-ERK, ERK, p-EGFR, cyclin D1 and GAPDH in AMPK WT and KO MEF cells. D, immunoblot analysis of cyclin D1, p-ERK, ERK, p-EGFR, AMPK, and GAPDH in KO MEF cells treated with vehicle (−), PD (PD98059, 20 μM) and AG (AG1478, 1 μM), and WT MEF cells. E, immunoblot analysis of p-ERK, ERK, p-EGFR, AMPK and GAPDH in WT and KO MEF cells treated with vehicle, AICAR (1 mM), or metformin (2 mM).

Article Snippet: Antibodies used included phospho-ERK (p-ERK), ERK, phospho-EGFR (p-EGFR), AMPK, ACC, DDB1, DDB2, XPC, E2F4, p130, Lamin B, β-actin, GAPDH (Santa Cruz), cyclin D1 (BD Bioscience) p-AMPK (T172) and p-ACC (S79)(Cell Signaling Technology).

Techniques: Western Blot

Journal: International Journal of Ophthalmology

Article Title: Inhibition of EGFR attenuates EGF-induced activation of retinal pigment epithelium cell via EGFR/AKT signaling pathway

doi: 10.18240/ijo.2024.06.05

Figure Lengend Snippet: ARPE-19 cells were stimulated by 100 ng/mL of EGF for 24h. A: The images of cells morphology were obtained after treatment, scale bar: 100 µm. B: Cellular viability was tested by the MTT assay, n=6. C, D: Cellular proliferation was analyzed by the BrdU labeling assay, scale bar: 50 µm, n=3. E, F: Cellular migration was measured by the wound healing assay, scale bar: 200 µm, n=3. G, H: After treatment with EGF (100 ng/mL) for 0, 0.25, 0.5, 1.0, 3.0, and 6.0h, EGFR, p-EGFR, AKT and p-AKT proteins in RPE cells were detected by Western blot assay. aP<0.05, bP<0.01. EGF: Epidermal growth factor; APRE-19: Adult retinalpigment epithelial cell line-19; EGFR: Epithelial growth factor receptor; AKT: Protein kinase B; MTT: Methyl thiazolyl tetrazolium; BrdU: 5-Bromodeoxyuridinc; DAPI: Diamidino-2-phenylindole.

Article Snippet: The EGFR, phosphorylated-EGFR (p-EGFR), AKT, phosphorylated-AKT (p-AKT), N-cadherin, α-smooth muscle actin (α-SMA), vimentin primary antibodies were from Cell Signaling Technology (Danvers, MA, USA).

Techniques: MTT Assay, Labeling, Migration, Wound Healing Assay, Western Blot

Journal: International Journal of Ophthalmology

Article Title: Inhibition of EGFR attenuates EGF-induced activation of retinal pigment epithelium cell via EGFR/AKT signaling pathway

doi: 10.18240/ijo.2024.06.05

Figure Lengend Snippet: A, B: ARPE-19 cells were treated with erlotinib (0, 5, 10, 20, and 50 µmol/L) for 24h. The expreesion of total EGFR and AKT proteins were measured by Western blot. aP<0.05, bP<0.01. C: After treatment with 50 µmol/L erlotinib for 24h, EGFR, F-actin, and nucleus in ARPE-19 cells were measured by immunofluorescence staining, scale bar: 50 µm. APRE-19: Adult retinal pigment epithelial cell line-19; EGFR: Epidermal growth factor receptor; AKT: Protein kinase B; DAPI: Diamidino-2-phenylindole.

Article Snippet: The EGFR, phosphorylated-EGFR (p-EGFR), AKT, phosphorylated-AKT (p-AKT), N-cadherin, α-smooth muscle actin (α-SMA), vimentin primary antibodies were from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Western Blot, Immunofluorescence, Staining

Journal: International Journal of Ophthalmology

Article Title: Inhibition of EGFR attenuates EGF-induced activation of retinal pigment epithelium cell via EGFR/AKT signaling pathway

doi: 10.18240/ijo.2024.06.05

Figure Lengend Snippet: ARPE-19 cells were pretreated by erlotinib (20 µmol/L) for 12h and then stimulated with 100 ng/mL of EGF for 24h. A, B: BrdU staining assay was used to test cellular proliferation, scale bar: 50 µm, n=3. C: Cellular viability was sized by MTT assay, n=8. D, E: Cellular migration was tested by the wound healing assay, scale bar: 200 µm, n=3. F, G: ARPE-19 cells were pretreated with 20 µmol/L erlotinib for 12h and then treated with EGF (100 ng/mL) for 15min. Western blotting was used to detected EGFR/AKT signaling pathway proteins expressions. H, I: After pretreatment with 20 µmol/L erlotinib for 12h, ARPE-19 cells were treated with EGF (100 ng/mL) for 24h. N-cadherin, α-SMA and vimentin proteins were determined by Western blot. aP<0.05, bP<0.01. EGF: Epidermal growth factor; MTT: Methyl thiazolyl tetrazolium; BrdU: 5-Bromodeoxyuridinc; APRE-19: Adult retinal pigment epithelial cell line-19; EGFR: Epidermal growth factor receptor; AKT: Protein kinase B; α-SMA: α-smooth muscle actin; DAPI: Diamidino-2-phenylindole.

Article Snippet: The EGFR, phosphorylated-EGFR (p-EGFR), AKT, phosphorylated-AKT (p-AKT), N-cadherin, α-smooth muscle actin (α-SMA), vimentin primary antibodies were from Cell Signaling Technology (Danvers, MA, USA).

Techniques: BrdU Staining, MTT Assay, Migration, Wound Healing Assay, Western Blot

Journal: International Journal of Ophthalmology

Article Title: Inhibition of EGFR attenuates EGF-induced activation of retinal pigment epithelium cell via EGFR/AKT signaling pathway

doi: 10.18240/ijo.2024.06.05

Figure Lengend Snippet: A, B: After EGFR-siRNA or control-siRNA were transfected into ARPE-19 cells, EGFR and AKT proteins were tested and analyzed by Western blotting. After EGFR-siRNA transfection, ARPE-19 cells were treated without or with 100 ng/mL EGF for 24h. C: Cellular viability was tested by MTT assay, n=8. D, E: Cellular proliferation was tested by the BrdU labeling assay, scale bar: 50 µm, n=3. F, G: Cellular migration was detected by the wound healing assay, scale bar: 200 µm, n=3. H, I: After transfected by control-siRNA or EGFR-siRNA, ARPE-19 cells were stimulated without or with 100 ng/mL of EGF for 15min. EGFR, p-EGFR, AKT and p-AKT protein were detected by Western blot. aP<0.05, bP<0.01. siRNA: Small interfering RNA; EGF: Epidermal growth factor; EGFR: Epidermal growth factor receptor; AKT: Protein kinase B; APRE-19: Adult retinal pigment epithelial cell line-19; MTT: Methyl thiazolyl tetrazolium; BrdU: 5-Bromodeoxyuridinc; DAPI: Diamidino-2-phenylindole.

Article Snippet: The EGFR, phosphorylated-EGFR (p-EGFR), AKT, phosphorylated-AKT (p-AKT), N-cadherin, α-smooth muscle actin (α-SMA), vimentin primary antibodies were from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Control, Transfection, Western Blot, MTT Assay, Labeling, Migration, Wound Healing Assay, Small Interfering RNA

Journal: International Journal of Ophthalmology

Article Title: Inhibition of EGFR attenuates EGF-induced activation of retinal pigment epithelium cell via EGFR/AKT signaling pathway

doi: 10.18240/ijo.2024.06.05

Figure Lengend Snippet: A, B: After EGFR-siRNA transfections, ARPE-19 cells were treated without or with 100 ng/mL of EGF for 24h. N-cadherin, α-SMA and vimentin proteins were detected by Western blot. aP<0.05, bP<0.01. EGF: Epidermal growth factor; EGFR: Epidermal growth factor receptor; EMT: Epithelial-mesenchymal transition; APRE-19: Adult retinal pigment epithelial cell line-19; siRNA: Small interfering RNA; α-SMA: α-smooth muscle actin.

Article Snippet: The EGFR, phosphorylated-EGFR (p-EGFR), AKT, phosphorylated-AKT (p-AKT), N-cadherin, α-smooth muscle actin (α-SMA), vimentin primary antibodies were from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Transfection, Western Blot, Small Interfering RNA