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Cell Signaling Technology Inc anti egfr
Proposed signaling amplifying mechanism in HCT-116 cells exposed to hyperthermia. At 37 °C, mutant KRAS supports ERK signaling, which in turn augments the already deregulated by a mutation WNT/beta-catenin activity. Signaling through <t>HGFR,</t> a transcriptional target of WNT/beta-catenin activity, feeds back positively the ERK and WNT pathways. At 42 °C, hyperthermia-induced EGF and WNT ligands allow for a switch from HGFR to <t>EGFR</t> signaling, and to increasing in time signaling amplifying mechanism.
Anti Egfr, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 27 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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1) Product Images from "In Hyperthermia Increased ERK and WNT Signaling Suppress Colorectal Cancer Cell Growth"

Article Title: In Hyperthermia Increased ERK and WNT Signaling Suppress Colorectal Cancer Cell Growth

Journal: Cancers

doi: 10.3390/cancers8050049

Proposed signaling amplifying mechanism in HCT-116 cells exposed to hyperthermia. At 37 °C, mutant KRAS supports ERK signaling, which in turn augments the already deregulated by a mutation WNT/beta-catenin activity. Signaling through HGFR, a transcriptional target of WNT/beta-catenin activity, feeds back positively the ERK and WNT pathways. At 42 °C, hyperthermia-induced EGF and WNT ligands allow for a switch from HGFR to EGFR signaling, and to increasing in time signaling amplifying mechanism.
Figure Legend Snippet: Proposed signaling amplifying mechanism in HCT-116 cells exposed to hyperthermia. At 37 °C, mutant KRAS supports ERK signaling, which in turn augments the already deregulated by a mutation WNT/beta-catenin activity. Signaling through HGFR, a transcriptional target of WNT/beta-catenin activity, feeds back positively the ERK and WNT pathways. At 42 °C, hyperthermia-induced EGF and WNT ligands allow for a switch from HGFR to EGFR signaling, and to increasing in time signaling amplifying mechanism.

Techniques Used: Mutagenesis, Activity Assay

2) Product Images from "Metabolic flux-driven sialylation alters internalization, recycling, and drug sensitivity of the epidermal growth factor receptor (EGFR) in SW1990 pancreatic cancer cells"

Article Title: Metabolic flux-driven sialylation alters internalization, recycling, and drug sensitivity of the epidermal growth factor receptor (EGFR) in SW1990 pancreatic cancer cells

Journal: Oncotarget

doi: 10.18632/oncotarget.11582

Western blot analysis of SW1990 cells treated with 50 μM 1,3,4- O -Bu 3 ManNAc, 30 nM of erlotinib, or both compounds in combination showed ( A ) EGFR levels that are not affected by the analog alone are decreased by erlotinib. ( B ) EGFR phosphorylation is inhibited by both compounds individually as well as in combination. ( C ) and ( D ) p-AKT and p-ERK1/2 levels, respectively, are not significantly affected by any of the treatment conditions. ( E ) p-STAT3 levels are inhibited by the analog but are not affected by erlotinib; however the combination of the two compounds leads to significantly lower p-STAT3 levels when compared to the analog alone. At least 3 biological replicates were carried out for each experiment with data expressed as mean ± standard error mean (SEM). * indicates a p value of
Figure Legend Snippet: Western blot analysis of SW1990 cells treated with 50 μM 1,3,4- O -Bu 3 ManNAc, 30 nM of erlotinib, or both compounds in combination showed ( A ) EGFR levels that are not affected by the analog alone are decreased by erlotinib. ( B ) EGFR phosphorylation is inhibited by both compounds individually as well as in combination. ( C ) and ( D ) p-AKT and p-ERK1/2 levels, respectively, are not significantly affected by any of the treatment conditions. ( E ) p-STAT3 levels are inhibited by the analog but are not affected by erlotinib; however the combination of the two compounds leads to significantly lower p-STAT3 levels when compared to the analog alone. At least 3 biological replicates were carried out for each experiment with data expressed as mean ± standard error mean (SEM). * indicates a p value of

Techniques Used: Western Blot

3) Product Images from "Luteolin inhibits Musashi1 binding to RNA and disrupts cancer phenotypes in glioblastoma cells"

Article Title: Luteolin inhibits Musashi1 binding to RNA and disrupts cancer phenotypes in glioblastoma cells

Journal: RNA Biology

doi: 10.1080/15476286.2018.1539607

Luteolin impacts the expression of Musashi1 target genes. a) Western blot showing the impact of luteolin treatment on the expression of Msi1 target genes (PDGFRα, IGF-IR, EGFR, CCND1 and CDK6) 48 hours post-treatment. Msi1 functions as a positive regulator of these transcripts by increasing their translation or stability. Tubulin was used as loading control. b) We used a luciferase reporter containing the 3’UTR of PDGFRα to corroborate that luteolin treatment disrupts Msi1 regulatory functions. Msi1 is a positive regulator of PDGFRα translation. HT1080 cells were co-transfected with plasmids expressing either GST or Msi1. Msi1 expression affected the expression of the reporter, resulting increased luciferase activity. The effect was largely diminished when luteolin was added after transfection. Experiments were done in triplicate. A representative western is shown. Statistical significance was calculated by multiple t test. All data are shown as means ± s.d. (*P
Figure Legend Snippet: Luteolin impacts the expression of Musashi1 target genes. a) Western blot showing the impact of luteolin treatment on the expression of Msi1 target genes (PDGFRα, IGF-IR, EGFR, CCND1 and CDK6) 48 hours post-treatment. Msi1 functions as a positive regulator of these transcripts by increasing their translation or stability. Tubulin was used as loading control. b) We used a luciferase reporter containing the 3’UTR of PDGFRα to corroborate that luteolin treatment disrupts Msi1 regulatory functions. Msi1 is a positive regulator of PDGFRα translation. HT1080 cells were co-transfected with plasmids expressing either GST or Msi1. Msi1 expression affected the expression of the reporter, resulting increased luciferase activity. The effect was largely diminished when luteolin was added after transfection. Experiments were done in triplicate. A representative western is shown. Statistical significance was calculated by multiple t test. All data are shown as means ± s.d. (*P

Techniques Used: Expressing, Western Blot, Luciferase, Transfection, Activity Assay

4) Product Images from "EGFR NUCLEAR TRANSLOCATION MODULATES DNA REPAIR FOLLOWING CISPLATIN AND IONIZING RADIATION TREATMENT"

Article Title: EGFR NUCLEAR TRANSLOCATION MODULATES DNA REPAIR FOLLOWING CISPLATIN AND IONIZING RADIATION TREATMENT

Journal: Cancer research

doi: 10.1158/0008-5472.CAN-10-2384

EGFR and DNAPKcs cellular localisation following IR treatment. NIH3T3 transiently transfected with wtEGFR, NLS123, L858R, LNLS123, EGFRvIII, M1, M12, KMT, ΔNLS and Vector control were serum starved for 24 hours, treated with 4Gy Ionising radiation and then fixed with 4%PFA 20 minutes following treatment. Cells were stained with Goat anti-Rabbit Alexa fluor 647 (EGFR), Goat anti-Mouse Alexa fluor 488 (DNAPKcs), and Dapi (nucleus).
Figure Legend Snippet: EGFR and DNAPKcs cellular localisation following IR treatment. NIH3T3 transiently transfected with wtEGFR, NLS123, L858R, LNLS123, EGFRvIII, M1, M12, KMT, ΔNLS and Vector control were serum starved for 24 hours, treated with 4Gy Ionising radiation and then fixed with 4%PFA 20 minutes following treatment. Cells were stained with Goat anti-Rabbit Alexa fluor 647 (EGFR), Goat anti-Mouse Alexa fluor 488 (DNAPKcs), and Dapi (nucleus).

Techniques Used: Transfection, Plasmid Preparation, Staining

(A) EGFR-DNAPKcs complex cellular localization. Stable NIH3T3 cells expressing wtEGFR, NLS123, EGFRvIII and Vector control were treated with 50μM cisplatin for one hour in serum free media and then fixed with 4%PFA 18 hours following treatment or 4 Gy and then fixed with 4%PFA 20 minutes follwowing radiation. Cells were then immuno blocked with anti-rabbit EGFR and anti-mouse DNAPKcs. Interacting complexes were then visulised via the duo link proximit assay. Each red spot represents a single interaction. (B) EGFR modulation of DNA-PK kinase activity. Stable NIH3T3 cells expressing wtEGFR, NLS123, L858R, LNLS123 and EGFRvIII were treated with 50μM cisplatin for one hour or 4gy or 100ng/ml EGF in serum free media. 18 hours following the treatment with cisplatin, 20 minutes following the treatment with IR and at 1 hour following EGF incubation samples prepared for the DNAPK Kinase assay. The graph shows the percentage change in DNAPK activity following each treatment compared to untreated. Stars indicate statistical significance.
Figure Legend Snippet: (A) EGFR-DNAPKcs complex cellular localization. Stable NIH3T3 cells expressing wtEGFR, NLS123, EGFRvIII and Vector control were treated with 50μM cisplatin for one hour in serum free media and then fixed with 4%PFA 18 hours following treatment or 4 Gy and then fixed with 4%PFA 20 minutes follwowing radiation. Cells were then immuno blocked with anti-rabbit EGFR and anti-mouse DNAPKcs. Interacting complexes were then visulised via the duo link proximit assay. Each red spot represents a single interaction. (B) EGFR modulation of DNA-PK kinase activity. Stable NIH3T3 cells expressing wtEGFR, NLS123, L858R, LNLS123 and EGFRvIII were treated with 50μM cisplatin for one hour or 4gy or 100ng/ml EGF in serum free media. 18 hours following the treatment with cisplatin, 20 minutes following the treatment with IR and at 1 hour following EGF incubation samples prepared for the DNAPK Kinase assay. The graph shows the percentage change in DNAPK activity following each treatment compared to untreated. Stars indicate statistical significance.

Techniques Used: Expressing, Plasmid Preparation, Activity Assay, Incubation, Kinase Assay

(A) wtEGFR Transfected NI3T3 cells were treated with 50μM cisplatin for 1 hour in serum free media. Cells were then lysed 1,9,12,15,18,24, hours following treatment. 750μg of protein lysate were immunoprecipitated using anti DNAPKcs and blotted with anti EGFR and anti DNAPKcs. EGFR pull-down was quantified by 2D densitometric analysis and shown as a binding fold compared to the untreated control. Mouse unrelated antibody (M IGg) was used as negative control. (B) Stable NIH3T3 cells expressing wtEGFR, NLS123, L858R, LNLS123, EGFRvIII, and Vector control were treated with 50μM cisplatin or 4 G IR, or treated with 100ng/ml EGF as described in the materials and methods. 1.5mg of protein lysate was then immunoprecipitated using anti-EGFR monoclonal antibody and blotted with anti DNAPKcs, anti-PY20 and anti-EGFR.
Figure Legend Snippet: (A) wtEGFR Transfected NI3T3 cells were treated with 50μM cisplatin for 1 hour in serum free media. Cells were then lysed 1,9,12,15,18,24, hours following treatment. 750μg of protein lysate were immunoprecipitated using anti DNAPKcs and blotted with anti EGFR and anti DNAPKcs. EGFR pull-down was quantified by 2D densitometric analysis and shown as a binding fold compared to the untreated control. Mouse unrelated antibody (M IGg) was used as negative control. (B) Stable NIH3T3 cells expressing wtEGFR, NLS123, L858R, LNLS123, EGFRvIII, and Vector control were treated with 50μM cisplatin or 4 G IR, or treated with 100ng/ml EGF as described in the materials and methods. 1.5mg of protein lysate was then immunoprecipitated using anti-EGFR monoclonal antibody and blotted with anti DNAPKcs, anti-PY20 and anti-EGFR.

Techniques Used: Transfection, Immunoprecipitation, Binding Assay, Negative Control, Expressing, Plasmid Preparation

EGFR and DNAPKcs cellular localisation following cisplatin treatment. NIH3T3 transiently transfected with wtEGFR, NLS123, L858R, LNLS123, EGFRvIII, M1, M12, KMT, ΔNLS and Vector control, were treated with 50μM cisplatin for one hour in serum free media and then fixed with 4%PFA 18 hours following treatment. Cells were stained with Goat anti-Rabbit Alexa fluor 647 (EGFR), Goat anti-Mouse Alexa fluor 488 (DNAPKcs), and Dapi (nucleus).
Figure Legend Snippet: EGFR and DNAPKcs cellular localisation following cisplatin treatment. NIH3T3 transiently transfected with wtEGFR, NLS123, L858R, LNLS123, EGFRvIII, M1, M12, KMT, ΔNLS and Vector control, were treated with 50μM cisplatin for one hour in serum free media and then fixed with 4%PFA 18 hours following treatment. Cells were stained with Goat anti-Rabbit Alexa fluor 647 (EGFR), Goat anti-Mouse Alexa fluor 488 (DNAPKcs), and Dapi (nucleus).

Techniques Used: Transfection, Plasmid Preparation, Staining

EGFR and DNAPKcs cellular localisation following cisplatin treatment. Stable NIH3T3 cells expressing wtEGFR, NL123, L858R, LNLS123, EGFRvIII, Vector control were treated with 50μM cisplatin for one hour in serum free media and then fixed with 4%PFA 18 hours following treatment. Cells were stained with Goat anti-Rabbit Alexa fluor 647 (EGFR), Goat anti-Mouse Alexa fluor 488 (DNAPKcs), and Dapi (nucleus).
Figure Legend Snippet: EGFR and DNAPKcs cellular localisation following cisplatin treatment. Stable NIH3T3 cells expressing wtEGFR, NL123, L858R, LNLS123, EGFRvIII, Vector control were treated with 50μM cisplatin for one hour in serum free media and then fixed with 4%PFA 18 hours following treatment. Cells were stained with Goat anti-Rabbit Alexa fluor 647 (EGFR), Goat anti-Mouse Alexa fluor 488 (DNAPKcs), and Dapi (nucleus).

Techniques Used: Expressing, Plasmid Preparation, Staining

EGFR and DNAPKcs cellular localisation following IR treatment. Stable NIH3T3 cells expressing wtEGFR, NL123, L858R, LNLS123, EGFRvIII, Vector control were serum starved for 24 hours, treated with 4gy Ionising radiation and then fixed with 4%PFA 20 minutes following treatment. Cells were stained with Goat anti-Rabbit Alexa fluor 647 (EGFR), Goat anti-Mouse Alexa fluor 488 (DNAPKcs), and Dapi (nucleus).
Figure Legend Snippet: EGFR and DNAPKcs cellular localisation following IR treatment. Stable NIH3T3 cells expressing wtEGFR, NL123, L858R, LNLS123, EGFRvIII, Vector control were serum starved for 24 hours, treated with 4gy Ionising radiation and then fixed with 4%PFA 20 minutes following treatment. Cells were stained with Goat anti-Rabbit Alexa fluor 647 (EGFR), Goat anti-Mouse Alexa fluor 488 (DNAPKcs), and Dapi (nucleus).

Techniques Used: Expressing, Plasmid Preparation, Staining

5) Product Images from "Phosphoproteomics Reveals HMGA1, a CK2 Substrate, as a Drug-Resistant Target in Non-Small Cell Lung Cancer"

Article Title: Phosphoproteomics Reveals HMGA1, a CK2 Substrate, as a Drug-Resistant Target in Non-Small Cell Lung Cancer

Journal: Scientific Reports

doi: 10.1038/srep44021

HMGA1 knockdown influences EGFR pathways. ( a ) Phopsho-kinase profiles in PC9/gef shLacZ and PC9/gef shC1-2 (HMGA1) obtained by human phospho-kinase array (R D Systems, ARY003). Each membrane contains kinase specific (for example, 1−21) and positive control (P) antibodies spotted in duplicate. ( b ) Relative phosphorylation level was quantified by normalizing the pixel density of each positive control and shown in percentage. ( c ) Model of the mechanism of HMGA1-knockdown to recover sensitivity to gefitinib in resistant NSCLC.
Figure Legend Snippet: HMGA1 knockdown influences EGFR pathways. ( a ) Phopsho-kinase profiles in PC9/gef shLacZ and PC9/gef shC1-2 (HMGA1) obtained by human phospho-kinase array (R D Systems, ARY003). Each membrane contains kinase specific (for example, 1−21) and positive control (P) antibodies spotted in duplicate. ( b ) Relative phosphorylation level was quantified by normalizing the pixel density of each positive control and shown in percentage. ( c ) Model of the mechanism of HMGA1-knockdown to recover sensitivity to gefitinib in resistant NSCLC.

Techniques Used: Positive Control

6) Product Images from "EGF Induced RET Inhibitor Resistance in CCDC6-RET Lung Cancer Cells"

Article Title: EGF Induced RET Inhibitor Resistance in CCDC6-RET Lung Cancer Cells

Journal: Yonsei Medical Journal

doi: 10.3349/ymj.2017.58.1.9

EGFR ligand reduced the sensitivity of LC-2/ad cells to RET inhibitors in vitro . CCDC6-RET lung cancer cells were sensitive to RET inhibitors sunitinib, E7080, vandetanib, and sorafenib. LC-2/ad cells were pretreated with or without EGF (100 ng/mL) or HGF (50 ng/mL) for 24 hours and incubated with several concentrations of RET inhibitors sunitinib, E7080, vandetanib, or sorafenib. Cell growth was measured after 72 hours by celltiter-glo luminescent cell viability assay kits (Promega). Data are the mean of three independent experiments, each in triplicate. Bars, standard deviation. HGF, hepatocyte growth factor; EGFR, epidermal growth factor receptor; RET, rearranged during transfection.
Figure Legend Snippet: EGFR ligand reduced the sensitivity of LC-2/ad cells to RET inhibitors in vitro . CCDC6-RET lung cancer cells were sensitive to RET inhibitors sunitinib, E7080, vandetanib, and sorafenib. LC-2/ad cells were pretreated with or without EGF (100 ng/mL) or HGF (50 ng/mL) for 24 hours and incubated with several concentrations of RET inhibitors sunitinib, E7080, vandetanib, or sorafenib. Cell growth was measured after 72 hours by celltiter-glo luminescent cell viability assay kits (Promega). Data are the mean of three independent experiments, each in triplicate. Bars, standard deviation. HGF, hepatocyte growth factor; EGFR, epidermal growth factor receptor; RET, rearranged during transfection.

Techniques Used: In Vitro, Incubation, Cell Viability Assay, Standard Deviation, Transfection

LC-2/ad, CCDC6-RET lung cancer cells were highly sensitive to RET inhibitors. Sunitinib (A), E7080 (B), Vandetanib (C), Sorafenib (D). EGF receptor (EGFR) ligand, EGF, or activated EGFR triggered resistance to RET inhibitors by transducing bypass survival signaling through ERK and AKT. LC-2/ad cells were treated with or without gefitinib (1 µM) and/or EGF (100 ng/mL) for 2 hours with or without RET inhibitor at half-maximum inhibitory concentration (IC 50 ) for 1 hour. Cells were lysed and indicated proteins were detected by western blots. Shown are representative of three independent experiments. EGFR, epidermal growth factor receptor; RET, rearranged during transfection.
Figure Legend Snippet: LC-2/ad, CCDC6-RET lung cancer cells were highly sensitive to RET inhibitors. Sunitinib (A), E7080 (B), Vandetanib (C), Sorafenib (D). EGF receptor (EGFR) ligand, EGF, or activated EGFR triggered resistance to RET inhibitors by transducing bypass survival signaling through ERK and AKT. LC-2/ad cells were treated with or without gefitinib (1 µM) and/or EGF (100 ng/mL) for 2 hours with or without RET inhibitor at half-maximum inhibitory concentration (IC 50 ) for 1 hour. Cells were lysed and indicated proteins were detected by western blots. Shown are representative of three independent experiments. EGFR, epidermal growth factor receptor; RET, rearranged during transfection.

Techniques Used: Concentration Assay, Western Blot, Transfection

Abrogation of EGF-triggered resistance to RET inhibitors by EGFR-TKI. Sunitinib (A), E7080 (B), Vandetanib (C), Sorafenib (D). LC-2/ad cells were incubated with or without gefitinib (1 µM) for 2 hours and/or EGF (100 ng/mL) for 24 hours with or without RET inhibitors at half-maximum inhibitory concentration (IC 50 ), with cell growth determined after 72 hours. Cell growth was measured by celltiter-glo luminescent cell viability assay kit (Promega). * p
Figure Legend Snippet: Abrogation of EGF-triggered resistance to RET inhibitors by EGFR-TKI. Sunitinib (A), E7080 (B), Vandetanib (C), Sorafenib (D). LC-2/ad cells were incubated with or without gefitinib (1 µM) for 2 hours and/or EGF (100 ng/mL) for 24 hours with or without RET inhibitors at half-maximum inhibitory concentration (IC 50 ), with cell growth determined after 72 hours. Cell growth was measured by celltiter-glo luminescent cell viability assay kit (Promega). * p

Techniques Used: Incubation, Concentration Assay, Cell Viability Assay

Reduction of RET -fusion lung cancer cell sensitivity to RET inhibitors by co-culture with HUVEC endothelial cells. EGFR-specific siRNAs were transfected into LC-2/ad cells. After 24 hours, cells were cultured with or without HUVEC cells. After 24 hours, cells were incubated with RET inhibitors sunitinib, E7080, vandetanib, or sorafenib) at half-maximum inhibitory concentration (IC 50 ) for kinase inhibitors for 72 hours. Cell growth was determined by celltiter-glo luminescent cell viability assay kit (Promega). * p
Figure Legend Snippet: Reduction of RET -fusion lung cancer cell sensitivity to RET inhibitors by co-culture with HUVEC endothelial cells. EGFR-specific siRNAs were transfected into LC-2/ad cells. After 24 hours, cells were cultured with or without HUVEC cells. After 24 hours, cells were incubated with RET inhibitors sunitinib, E7080, vandetanib, or sorafenib) at half-maximum inhibitory concentration (IC 50 ) for kinase inhibitors for 72 hours. Cell growth was determined by celltiter-glo luminescent cell viability assay kit (Promega). * p

Techniques Used: Co-Culture Assay, Transfection, Cell Culture, Incubation, Concentration Assay, Cell Viability Assay

7) Product Images from "Sialidase NEU4 is involved in glioblastoma stem cell survival"

Article Title: Sialidase NEU4 is involved in glioblastoma stem cell survival

Journal: Cell Death & Disease

doi: 10.1038/cddis.2014.349

Effects of NEU4 silencing on signalling pathways related to stemness. ( a ) Western blot analysis of P-GSK-3 β (Ser9) in mock and iNEU4 U-GSCs. β -Actin was used as loading control. ( b ) Real-time PCR analysis of SHH , PTCH1 , GLI-1 , β catenin, axin 2 , and ( c ) NANOG , OCT-4 , SOX-2 , and CD133 in mock and iNEU4 U-GSCs. ( d ) Western blot analysis of EGFR in mock and iNEU4 U-GSCs. β -Actin was used as loading control. Values are the mean±S.D. of five independent experiments. Significance is based on the Student's t -test: * P
Figure Legend Snippet: Effects of NEU4 silencing on signalling pathways related to stemness. ( a ) Western blot analysis of P-GSK-3 β (Ser9) in mock and iNEU4 U-GSCs. β -Actin was used as loading control. ( b ) Real-time PCR analysis of SHH , PTCH1 , GLI-1 , β catenin, axin 2 , and ( c ) NANOG , OCT-4 , SOX-2 , and CD133 in mock and iNEU4 U-GSCs. ( d ) Western blot analysis of EGFR in mock and iNEU4 U-GSCs. β -Actin was used as loading control. Values are the mean±S.D. of five independent experiments. Significance is based on the Student's t -test: * P

Techniques Used: Western Blot, Real-time Polymerase Chain Reaction

8) Product Images from "EGF Potentiation of VEGF Production Is Cell Density Dependent in H292 EGFR Wild Type NSCLC Cell Line"

Article Title: EGF Potentiation of VEGF Production Is Cell Density Dependent in H292 EGFR Wild Type NSCLC Cell Line

Journal: International Journal of Molecular Sciences

doi: 10.3390/ijms151017686

( A ) Apparent increase in hepatocyte growth factor receptor (cMet) and epidermal growth factor receptor (EGFR) expression in H292 cells seeded at confluence compared to subconfluent H292 cells. Green = cMet, Orange = EGFR, Blue = Hoechst; and ( B ) Unaltered cMet ( left ) and EGFR ( right ) whole cell protein expression as measured by Meso Scale Discovery kit (MSD).
Figure Legend Snippet: ( A ) Apparent increase in hepatocyte growth factor receptor (cMet) and epidermal growth factor receptor (EGFR) expression in H292 cells seeded at confluence compared to subconfluent H292 cells. Green = cMet, Orange = EGFR, Blue = Hoechst; and ( B ) Unaltered cMet ( left ) and EGFR ( right ) whole cell protein expression as measured by Meso Scale Discovery kit (MSD).

Techniques Used: Expressing

EGFR compartmentalization in subconfluent vs . confluent cells. Far left, Orange = EGFR; Blue = Hoechst. ( A ) Separation of cytosolic ( center ) vs . extracellular membrane ( right ) staining of EGFR in confluent H292 cells; ( B ) Separation of cytosolic ( center ) vs . extracellular membrane ( right ) staining of EGFR in subconfluent H292 cells, various colors on center and right images indicate different objects/cells and ( C ) Membrane, cytosol staining ratio for cMet ( left ) and EGFR ( right ) in subconfluent and confluent H292 cells. * p
Figure Legend Snippet: EGFR compartmentalization in subconfluent vs . confluent cells. Far left, Orange = EGFR; Blue = Hoechst. ( A ) Separation of cytosolic ( center ) vs . extracellular membrane ( right ) staining of EGFR in confluent H292 cells; ( B ) Separation of cytosolic ( center ) vs . extracellular membrane ( right ) staining of EGFR in subconfluent H292 cells, various colors on center and right images indicate different objects/cells and ( C ) Membrane, cytosol staining ratio for cMet ( left ) and EGFR ( right ) in subconfluent and confluent H292 cells. * p

Techniques Used: Staining

9) Product Images from "Migration of FGF7-stimulated epithelial cells and VEGF-A-stimulated HUVECs depends on EGFR transactivation by ADAM17"

Article Title: Migration of FGF7-stimulated epithelial cells and VEGF-A-stimulated HUVECs depends on EGFR transactivation by ADAM17

Journal: Nature communications

doi: 10.1038/ncomms1232

A model for transactivation of the EGFR/ERK1/2 signaling pathway by FGF7/FGFR2b
Figure Legend Snippet: A model for transactivation of the EGFR/ERK1/2 signaling pathway by FGF7/FGFR2b

Techniques Used:

10) Product Images from "Activation of tyrosine kinases by mutation of the gatekeeper threonine"

Article Title: Activation of tyrosine kinases by mutation of the gatekeeper threonine

Journal: Nature structural & molecular biology

doi: 10.1038/nsmb.1486

Kinase activation and BAF3 cellular transformation by gatekeeper residue mutation of receptor tyrosine kinases. ( a ) Immunoblot analysis of BaF3 cells expressing gatekeeper mutants of PDGFRB. Above, total cell lysates probed with anti-phosphotyrosine antibody (anti-PY). Blots were stripped and reprobed with anti-PDGFRB antibody and anti–PDGFRB-phosphotyrosine-751 (anti-PY751). Below, immunoblots of Ni-NTA–purified histidine-tagged PDGFRB proteins probed with anti-PY, followed by stripping and reprobing with anti-PDGFRB antibody and anti-PY751. ( b ) Immunoblot analysis of BaF3 cells expressing gatekeeper mutants of PDGFRA. Above, total cell lysates probed with anti-PY. Blots were stripped and reprobed with anti-PDGFRA antibody and anti–PDGFRA-phosphotyrosine-754 (anti-PY754). Below, immunoblots of Ni-NTA–purified histidine-tagged PDGFRA proteins probed with anti-PY, followed by stripping and reprobing with anti-PDGFRA antibody and anti-PY754. ( c ) Immunoblot analysis of BaF3 cells expressing gatekeeper mutants of EGFR. Above, total cell lysates probed with anti-PY. Blots were stripped and reprobed with anti-EGFR antibody and anti–EGFR-phosphotyrosine-845 (anti-PY845). Below, immunoblots of Ni-NTA–purified histidine-tagged EGFR proteins probed with anti-PY, followed by stripping and reprobing with anti-EGFR antibody and anti-PY845. ( d – f ) Cell-proliferation assays of BaF3 cells expressing wild-type and gatekeeper mutants of PDGFRB ( d ), PDGFRA ( e ) and EGFR ( f ). Cells were plated in quadruplicate in 96-well plates at a density of 5,000 cells per well in the absence of IL-3 and scored when the wells became confluent.
Figure Legend Snippet: Kinase activation and BAF3 cellular transformation by gatekeeper residue mutation of receptor tyrosine kinases. ( a ) Immunoblot analysis of BaF3 cells expressing gatekeeper mutants of PDGFRB. Above, total cell lysates probed with anti-phosphotyrosine antibody (anti-PY). Blots were stripped and reprobed with anti-PDGFRB antibody and anti–PDGFRB-phosphotyrosine-751 (anti-PY751). Below, immunoblots of Ni-NTA–purified histidine-tagged PDGFRB proteins probed with anti-PY, followed by stripping and reprobing with anti-PDGFRB antibody and anti-PY751. ( b ) Immunoblot analysis of BaF3 cells expressing gatekeeper mutants of PDGFRA. Above, total cell lysates probed with anti-PY. Blots were stripped and reprobed with anti-PDGFRA antibody and anti–PDGFRA-phosphotyrosine-754 (anti-PY754). Below, immunoblots of Ni-NTA–purified histidine-tagged PDGFRA proteins probed with anti-PY, followed by stripping and reprobing with anti-PDGFRA antibody and anti-PY754. ( c ) Immunoblot analysis of BaF3 cells expressing gatekeeper mutants of EGFR. Above, total cell lysates probed with anti-PY. Blots were stripped and reprobed with anti-EGFR antibody and anti–EGFR-phosphotyrosine-845 (anti-PY845). Below, immunoblots of Ni-NTA–purified histidine-tagged EGFR proteins probed with anti-PY, followed by stripping and reprobing with anti-EGFR antibody and anti-PY845. ( d – f ) Cell-proliferation assays of BaF3 cells expressing wild-type and gatekeeper mutants of PDGFRB ( d ), PDGFRA ( e ) and EGFR ( f ). Cells were plated in quadruplicate in 96-well plates at a density of 5,000 cells per well in the absence of IL-3 and scored when the wells became confluent.

Techniques Used: Activation Assay, Transformation Assay, Mutagenesis, Expressing, Western Blot, Purification, Stripping Membranes

11) Product Images from "Berberine Inhibits Proliferation and Down-Regulates Epidermal Growth Factor Receptor through Activation of Cbl in Colon Tumor Cells"

Article Title: Berberine Inhibits Proliferation and Down-Regulates Epidermal Growth Factor Receptor through Activation of Cbl in Colon Tumor Cells

Journal: PLoS ONE

doi: 10.1371/journal.pone.0056666

Berberine down-regulates EGFR level and activates Cbl in colon tumor cells. (A) cells were cultured in serum-starved RPMI 1640 medium at 37°C (non-permissive condition) for 24 hours with or without berberine at 50 µM treatment for indicated times from the end of the experiment. Cellular lysates were collected for Western blot analysis to detect indicated signaling pathways. (B) cells were treated with berberine at 50 µM under non-permissive condition for 18 hours in the presence or absence of EGF (30 ng/ml) treatment for 5 minutes. 1 mg of cellular lysates were prepared for immunoprecipitation using an anti-EGFR antibody. EGFR and co-immunoprecipated proteins were analyzed using Western blot analysis. The β-actin blot was used as a protein loading control. (C) mRNA was isolated from cells cultured under non-permissive condition for 24 hours with or without berberine at 50 µM treatment for indicated times from the end of the experiment. Real-time PCR analysis was performed to detect the EGFR mRNA level. The EGFR mRNA expression level in the control group was set as 100%, and mRNA expression levels in treated groups were compared to the control group. Data in this Figure are representative of three separate experiments.
Figure Legend Snippet: Berberine down-regulates EGFR level and activates Cbl in colon tumor cells. (A) cells were cultured in serum-starved RPMI 1640 medium at 37°C (non-permissive condition) for 24 hours with or without berberine at 50 µM treatment for indicated times from the end of the experiment. Cellular lysates were collected for Western blot analysis to detect indicated signaling pathways. (B) cells were treated with berberine at 50 µM under non-permissive condition for 18 hours in the presence or absence of EGF (30 ng/ml) treatment for 5 minutes. 1 mg of cellular lysates were prepared for immunoprecipitation using an anti-EGFR antibody. EGFR and co-immunoprecipated proteins were analyzed using Western blot analysis. The β-actin blot was used as a protein loading control. (C) mRNA was isolated from cells cultured under non-permissive condition for 24 hours with or without berberine at 50 µM treatment for indicated times from the end of the experiment. Real-time PCR analysis was performed to detect the EGFR mRNA level. The EGFR mRNA expression level in the control group was set as 100%, and mRNA expression levels in treated groups were compared to the control group. Data in this Figure are representative of three separate experiments.

Techniques Used: Cell Culture, Western Blot, Immunoprecipitation, Isolation, Real-time Polymerase Chain Reaction, Expressing

12) Product Images from "Pituitary Adenylate Cyclase-Activating Polypeptide Causes Tyrosine Phosphorylation of the Epidermal Growth Factor Receptor in Lung Cancer Cells"

Article Title: Pituitary Adenylate Cyclase-Activating Polypeptide Causes Tyrosine Phosphorylation of the Epidermal Growth Factor Receptor in Lung Cancer Cells

Journal: The Journal of Pharmacology and Experimental Therapeutics

doi: 10.1124/jpet.111.190033

PACAP-27 increases EGFR and ERK tyrosine phosphorylation in a time- and dose-dependent manner. A, the ability of 100 nM PACAP-27 to increase EGFR tyrosine phosphorylation was investigated as a function of time by using NCI-H838 cells and antiphospho-Tyr-1068-EGFR antibody. B, the mean percentage of EGFR tyrosine phosphorylation ± S.D is indicated for three determinations. C, the ability of 100 nM PACAP-27 to increase ERK tyrosine phosphorylation is indicated by using NCI-H838 cells. D, the mean percentage of ERK tyrosine phosphorylation ± S.D is indicated for four determinations. E, the ability of PACAP-27 to increase EGFR tyrosine phosphorylation is indicated as a function of dose by using NCI-H838 cells. F, the mean percentage of EGFR tyrosine phosphorylation ± S.D is indicated for four determinations. **, p
Figure Legend Snippet: PACAP-27 increases EGFR and ERK tyrosine phosphorylation in a time- and dose-dependent manner. A, the ability of 100 nM PACAP-27 to increase EGFR tyrosine phosphorylation was investigated as a function of time by using NCI-H838 cells and antiphospho-Tyr-1068-EGFR antibody. B, the mean percentage of EGFR tyrosine phosphorylation ± S.D is indicated for three determinations. C, the ability of 100 nM PACAP-27 to increase ERK tyrosine phosphorylation is indicated by using NCI-H838 cells. D, the mean percentage of ERK tyrosine phosphorylation ± S.D is indicated for four determinations. E, the ability of PACAP-27 to increase EGFR tyrosine phosphorylation is indicated as a function of dose by using NCI-H838 cells. F, the mean percentage of EGFR tyrosine phosphorylation ± S.D is indicated for four determinations. **, p

Techniques Used:

13) Product Images from "Downregulation of ER-α36 expression sensitizes HER2 overexpressing breast cancer cells to tamoxifen"

Article Title: Downregulation of ER-α36 expression sensitizes HER2 overexpressing breast cancer cells to tamoxifen

Journal: American Journal of Cancer Research

doi:

Disruption of the ER-α36-EGFR/HER2 positive regulatory loops sensitizes HER2 expressing breast cancer stem/progenitor cells to tamoxifen. (A) Western blot analysis of the expression of ER-α36, ER-α66, EGFR and HER2 in the monolayer cells grown on attachment dishes (A) and tumorsphere cells grown on low-attachment dishes (T). Band density (% of β-actin) is also shown. (B C) Tumorsphere cells derived from BT474 and MCF7/HER2-18 cells were treated with 5 μM of Broussoflavonol B (BB) or Lapatinib (LAP) for five days. Western blot analysis of expression levels different proteins was performed. (D) Tumorsphere formation assay was used to assess the effects of tamoxifen alone or together with Lapatinib (LAP) or Broussoflavonol B (BB) on the breast cancer stem/progenitor cells derived from MCF7/HER2-18 cells. The representative results are shown. (E) The numbers of tumorspheres formed by the cells treated with tamoxifen alone or together with Lapatinib (LAP) or Broussoflavonol B (BB). (F) Tumorsphere formation assay was used to assess the effects of tamoxifen alone or together with Lapatinib (LAP) or Broussoflavonol B (BB) on the breast cancer stem/progenitor cells derived from BT474 cells. The representative results are shown. (G) The numbers of tumorspheres formed by the cells treated with tamoxifen alone or together with Lapatinib (LAP) or Broussoflavonol B (BB). The columns represent the means of three experiments; bars, SE. * #, P
Figure Legend Snippet: Disruption of the ER-α36-EGFR/HER2 positive regulatory loops sensitizes HER2 expressing breast cancer stem/progenitor cells to tamoxifen. (A) Western blot analysis of the expression of ER-α36, ER-α66, EGFR and HER2 in the monolayer cells grown on attachment dishes (A) and tumorsphere cells grown on low-attachment dishes (T). Band density (% of β-actin) is also shown. (B C) Tumorsphere cells derived from BT474 and MCF7/HER2-18 cells were treated with 5 μM of Broussoflavonol B (BB) or Lapatinib (LAP) for five days. Western blot analysis of expression levels different proteins was performed. (D) Tumorsphere formation assay was used to assess the effects of tamoxifen alone or together with Lapatinib (LAP) or Broussoflavonol B (BB) on the breast cancer stem/progenitor cells derived from MCF7/HER2-18 cells. The representative results are shown. (E) The numbers of tumorspheres formed by the cells treated with tamoxifen alone or together with Lapatinib (LAP) or Broussoflavonol B (BB). (F) Tumorsphere formation assay was used to assess the effects of tamoxifen alone or together with Lapatinib (LAP) or Broussoflavonol B (BB) on the breast cancer stem/progenitor cells derived from BT474 cells. The representative results are shown. (G) The numbers of tumorspheres formed by the cells treated with tamoxifen alone or together with Lapatinib (LAP) or Broussoflavonol B (BB). The columns represent the means of three experiments; bars, SE. * #, P

Techniques Used: Expressing, Western Blot, Derivative Assay, Tube Formation Assay

HER2 expressing breast cancer cells exhibit enhanced expression of ER-α36 and EGFR as well as tamoxifen resistance. A B. Western blot analysis of the expression levels of ER-α66, ER-α36, HER2 and EGFR in ER-positive breast cancer MCF7, BT474 and MCF7HER2-18 cells. C. Cells were treated with indicated concentrations of tamoxifen for seven days and the numbers of survived cells were counted. Each point represents the means of three experiments.
Figure Legend Snippet: HER2 expressing breast cancer cells exhibit enhanced expression of ER-α36 and EGFR as well as tamoxifen resistance. A B. Western blot analysis of the expression levels of ER-α66, ER-α36, HER2 and EGFR in ER-positive breast cancer MCF7, BT474 and MCF7HER2-18 cells. C. Cells were treated with indicated concentrations of tamoxifen for seven days and the numbers of survived cells were counted. Each point represents the means of three experiments.

Techniques Used: Expressing, Western Blot

ER-α36 disruptor Broussoflavonol B restores tamoxifen sensitivity in HER2 expressing cells. A C. Western blot analysis of the expression of ER-α36, ER-α66, EGFR and HER2 in MCF7 and MCF7/HER2-18 and BT474 cells treated with indicated concentrations of Broussoflavonol B (BB) for 12 hours. B D. Cells were treated with indicated concentrations of tamoxifen (TAM) together with vehicle or 1 μM of Broussoflavonol B (BB) for seven days and the numbers of survived cells were counted. The columns represent the means of three experiments; bars, SE. * and #, P
Figure Legend Snippet: ER-α36 disruptor Broussoflavonol B restores tamoxifen sensitivity in HER2 expressing cells. A C. Western blot analysis of the expression of ER-α36, ER-α66, EGFR and HER2 in MCF7 and MCF7/HER2-18 and BT474 cells treated with indicated concentrations of Broussoflavonol B (BB) for 12 hours. B D. Cells were treated with indicated concentrations of tamoxifen (TAM) together with vehicle or 1 μM of Broussoflavonol B (BB) for seven days and the numbers of survived cells were counted. The columns represent the means of three experiments; bars, SE. * and #, P

Techniques Used: Expressing, Western Blot

Dual kinase inhibitor Lapatinib downregulates ER-α36 expression and sensitizes HER2 expressing cells to tamoxifen. A. Western blot analysis of the expression of phosphorylated EGFR and HER2 in MCF7/HER2-18 treated with indicated concentrations of Lapatinib for 12 hours. B. Western blot analysis of the expression of phosphorylated EGFR and HER2 in BT474 cells treated with indicated concentrations of Lapatinib for 12 hours. C E. Western blot analysis of the expression of ER-α36 and ER-α66 as well as EGFR and HER2 in parental MCF7, MCF7/HER2-18 and BT474 cells treated with vehicle or indicated concentrations of Lapatinib (LAP) for 12 hours. D F. Cells were treated with indicated concentrations of tamoxifen (TAM) together with vehicle or 1 μM of Lapatinib (LAP) for seven days and the numbers of survived cells were counted. The columns represent the means of three experiments; bars, SE. * and #, P
Figure Legend Snippet: Dual kinase inhibitor Lapatinib downregulates ER-α36 expression and sensitizes HER2 expressing cells to tamoxifen. A. Western blot analysis of the expression of phosphorylated EGFR and HER2 in MCF7/HER2-18 treated with indicated concentrations of Lapatinib for 12 hours. B. Western blot analysis of the expression of phosphorylated EGFR and HER2 in BT474 cells treated with indicated concentrations of Lapatinib for 12 hours. C E. Western blot analysis of the expression of ER-α36 and ER-α66 as well as EGFR and HER2 in parental MCF7, MCF7/HER2-18 and BT474 cells treated with vehicle or indicated concentrations of Lapatinib (LAP) for 12 hours. D F. Cells were treated with indicated concentrations of tamoxifen (TAM) together with vehicle or 1 μM of Lapatinib (LAP) for seven days and the numbers of survived cells were counted. The columns represent the means of three experiments; bars, SE. * and #, P

Techniques Used: Expressing, Western Blot

Knock-down of ER-α36 expression sensitizes HER2 expressing cells to tamoxifen. A. Western blot analysis of the expression levels of ER-α66, ER-α36, HER2 and EGFR in MCF7/HER2-18 cells transfected with an empty expression vector (MCF7/HER2-18/V) and with the ER-α36 shRNA expression vector (MCF7/HER2-18/Si36). B. Western blot analysis of the expression levels of ER-α66, ER-α36, HER2 and EGFR in BT474 cells transfected with an empty expression vector (BT474/V) and with the ER-α36 shRNA expression vector (BT474/Si36). C D. Cells were treated with indicated concentrations of tamoxifen for seven days and the numbers of survived cells were counted. The columns represent the means of three experiments; bars, SE. * and #, P
Figure Legend Snippet: Knock-down of ER-α36 expression sensitizes HER2 expressing cells to tamoxifen. A. Western blot analysis of the expression levels of ER-α66, ER-α36, HER2 and EGFR in MCF7/HER2-18 cells transfected with an empty expression vector (MCF7/HER2-18/V) and with the ER-α36 shRNA expression vector (MCF7/HER2-18/Si36). B. Western blot analysis of the expression levels of ER-α66, ER-α36, HER2 and EGFR in BT474 cells transfected with an empty expression vector (BT474/V) and with the ER-α36 shRNA expression vector (BT474/Si36). C D. Cells were treated with indicated concentrations of tamoxifen for seven days and the numbers of survived cells were counted. The columns represent the means of three experiments; bars, SE. * and #, P

Techniques Used: Expressing, Western Blot, Transfection, Plasmid Preparation, shRNA

14) Product Images from "The role of PTRF/Cavin1 as a biomarker in both glioma and serum exosomes"

Article Title: The role of PTRF/Cavin1 as a biomarker in both glioma and serum exosomes

Journal: Theranostics

doi: 10.7150/thno.22952

B. The scale bar corresponds to 20 μm. (H) Ten micrograms of exosomes isolated from the supernatants of U87, U251 and TBD0313 cells transduced with EGFRvIII were treated with 10 μl of proteinase K or PBS for 10 min. Western blot analysis showed that EGFRvIII, EGFR and PTRF were totally degraded, while CD63 was partly intact. (I) Cartoon showing EGFRvIII, EGFR and PTRF expression on exosome membranes and CD63 expression on both exosome membranes and inside exosomes.
Figure Legend Snippet: B. The scale bar corresponds to 20 μm. (H) Ten micrograms of exosomes isolated from the supernatants of U87, U251 and TBD0313 cells transduced with EGFRvIII were treated with 10 μl of proteinase K or PBS for 10 min. Western blot analysis showed that EGFRvIII, EGFR and PTRF were totally degraded, while CD63 was partly intact. (I) Cartoon showing EGFRvIII, EGFR and PTRF expression on exosome membranes and CD63 expression on both exosome membranes and inside exosomes.

Techniques Used: Isolation, Transduction, Western Blot, Expressing

Schematic mechanism for intercellular communication via exosomes. PTRF is a downstream effector of the EGFR/PI3K/AKT pathway via the H3K4me3 and H3K27me3 modifications. Exosomes from GBM cells delivered biological components, such as EGFRvIII, PTRF and EGFRvIII mRNA, to nearby cells and induced their malignancy. Exosomes could also be released into the blood and serve as a detectable biomarker.
Figure Legend Snippet: Schematic mechanism for intercellular communication via exosomes. PTRF is a downstream effector of the EGFR/PI3K/AKT pathway via the H3K4me3 and H3K27me3 modifications. Exosomes from GBM cells delivered biological components, such as EGFRvIII, PTRF and EGFRvIII mRNA, to nearby cells and induced their malignancy. Exosomes could also be released into the blood and serve as a detectable biomarker.

Techniques Used: Biomarker Assay

PTRF expression is positively associated with EGFR and EGFRvIII. (A) After stably overexpressing EGFRvIII, DIA proteomic methods were used to evaluate total protein expression. Among these proteins, PTRF expression was increased. (B) GBM cell lines were either stimulated by EGF or transduced with EGFRvIII for 48 hours, and the expression of EGFRvIII, PTRF and GAPDH was evaluated by western blot. GAPDH served as the negative control. (C) RT-qPCR experiments were performed to detect PTRF mRNA expression. After EGF stimulation and EGFRvIII transduction, PTRF mRNA expression was gently increased. Error bars in the RT-qPCR results indicate the standard error of the mean. Significant differences in the experimental groups were compared with those of the control group (**P
Figure Legend Snippet: PTRF expression is positively associated with EGFR and EGFRvIII. (A) After stably overexpressing EGFRvIII, DIA proteomic methods were used to evaluate total protein expression. Among these proteins, PTRF expression was increased. (B) GBM cell lines were either stimulated by EGF or transduced with EGFRvIII for 48 hours, and the expression of EGFRvIII, PTRF and GAPDH was evaluated by western blot. GAPDH served as the negative control. (C) RT-qPCR experiments were performed to detect PTRF mRNA expression. After EGF stimulation and EGFRvIII transduction, PTRF mRNA expression was gently increased. Error bars in the RT-qPCR results indicate the standard error of the mean. Significant differences in the experimental groups were compared with those of the control group (**P

Techniques Used: Expressing, Stable Transfection, Transduction, Western Blot, Negative Control, Quantitative RT-PCR

15) Product Images from "Tyrosine kinase receptor EGFR regulates the switch in cancer cells between cell survival and cell death induced by autophagy in hypoxia"

Article Title: Tyrosine kinase receptor EGFR regulates the switch in cancer cells between cell survival and cell death induced by autophagy in hypoxia

Journal: Autophagy

doi: 10.1080/15548627.2016.1164357

Hypoxia increases EGFR expression but inhibits its tyrosine phosphorylation. (A) The total protein level of EGFR and the activation of its tyrosine kinase, as represented by the phosphorylation of EGFR at tyrosine1068 (P-EGFR [Y1068]), was determined
Figure Legend Snippet: Hypoxia increases EGFR expression but inhibits its tyrosine phosphorylation. (A) The total protein level of EGFR and the activation of its tyrosine kinase, as represented by the phosphorylation of EGFR at tyrosine1068 (P-EGFR [Y1068]), was determined

Techniques Used: Expressing, Activation Assay

Knockdown of EGFR shifts autophagy-induced cell survival to autophagy-induced cell death at an early time in hypoxia. (A) U87 cells were knocked down for EGFR and ATG5 genes confirmed by western blotting. (B) Cells with knockdown of EGFR and ATG5 genes
Figure Legend Snippet: Knockdown of EGFR shifts autophagy-induced cell survival to autophagy-induced cell death at an early time in hypoxia. (A) U87 cells were knocked down for EGFR and ATG5 genes confirmed by western blotting. (B) Cells with knockdown of EGFR and ATG5 genes

Techniques Used: Western Blot

Activation of EGFR tyrosine kinase regulates the binding of BCL2 to BECN1 in hypoxia. (A) BCL2 protein level over a 72 h time course in hypoxia was determined by western blot in U87 and A549 cells. ACTB was used as a loading control. (B) BCL2
Figure Legend Snippet: Activation of EGFR tyrosine kinase regulates the binding of BCL2 to BECN1 in hypoxia. (A) BCL2 protein level over a 72 h time course in hypoxia was determined by western blot in U87 and A549 cells. ACTB was used as a loading control. (B) BCL2

Techniques Used: Activation Assay, Binding Assay, Western Blot

16) Product Images from "FoxM1 drives ADAM17/EGFR activation loop to promote mesenchymal transition in glioblastoma"

Article Title: FoxM1 drives ADAM17/EGFR activation loop to promote mesenchymal transition in glioblastoma

Journal: Cell Death & Disease

doi: 10.1038/s41419-018-0482-4

FoxM1/ADAM17 axis drives EGFR/AKT/GSK3β signaling and maintains FoxM1 stability in glioma cells. a Heatmap revealed that the expression of FoxM1 and ADAM17 were correlated with that of EGFR in glioblastoma samples from TCGA database. b , c The effects of FoxM1 on EGFR/AKT/GSK3β signaling pathway in glioma cells were investigated by western blot. d The key components of EGFR/AKT/GSK3β signaling were detected after knocking down FoxM1 and overexpressing ADAM17 by western blot in U251MG and U87MG cells. e The effects of FoxM1 upregulation and ADAM17 knockdown on EGFR/AKT/GSK3β signaling pathway were confirmed by western blot in SW1783 and LN229 cells. f U87MG cells were treated with TAPI-2(ADAM17 inhibitor), and then indicated proteins were measured by western blot. g After transfected with sh-ADAM17, U87MG cells were treated with CHX (0, 2, 4, 6 h) and then western blot was used to determine the expression of FoxM1. h FoxM1 levels were measured in MEF GSK3β+/+ and GSK3β−/− cells with CHX (0, 2, 4, 6 h) . i The effects of ADAM17 downregulation on FoxM1 expression in MEF GSK3β+/+ and GSK3β−/− cells. j The levels of indicated proteins were detected by western blot in U87MG-sh-ADAM17 cells treated with CHX and LiCl (0, 2, 4, 6 h)
Figure Legend Snippet: FoxM1/ADAM17 axis drives EGFR/AKT/GSK3β signaling and maintains FoxM1 stability in glioma cells. a Heatmap revealed that the expression of FoxM1 and ADAM17 were correlated with that of EGFR in glioblastoma samples from TCGA database. b , c The effects of FoxM1 on EGFR/AKT/GSK3β signaling pathway in glioma cells were investigated by western blot. d The key components of EGFR/AKT/GSK3β signaling were detected after knocking down FoxM1 and overexpressing ADAM17 by western blot in U251MG and U87MG cells. e The effects of FoxM1 upregulation and ADAM17 knockdown on EGFR/AKT/GSK3β signaling pathway were confirmed by western blot in SW1783 and LN229 cells. f U87MG cells were treated with TAPI-2(ADAM17 inhibitor), and then indicated proteins were measured by western blot. g After transfected with sh-ADAM17, U87MG cells were treated with CHX (0, 2, 4, 6 h) and then western blot was used to determine the expression of FoxM1. h FoxM1 levels were measured in MEF GSK3β+/+ and GSK3β−/− cells with CHX (0, 2, 4, 6 h) . i The effects of ADAM17 downregulation on FoxM1 expression in MEF GSK3β+/+ and GSK3β−/− cells. j The levels of indicated proteins were detected by western blot in U87MG-sh-ADAM17 cells treated with CHX and LiCl (0, 2, 4, 6 h)

Techniques Used: Expressing, Western Blot, Transfection

17) Product Images from "Specific Inhibition of Tumor Cells by Oncogenic EGFR Specific Silencing by RNA interference"

Article Title: Specific Inhibition of Tumor Cells by Oncogenic EGFR Specific Silencing by RNA interference

Journal: PLoS ONE

doi: 10.1371/journal.pone.0073214

Oncogenic allele-specific RNAi. ( A ) Specific inhibition of mutant EGFR reporter alleles by ASP-RNAi. The effects of allele-specific siRNAs, si747/49_3D8 and si746/50_3D4, on expression of the target L747_E749del, A750P and E746_A750del EGFR mutant reporter alleles, respectively, and of the normal reporter alleles were examined using IC50 analysis (details in Methods). The IC50 values of the siRNAs for inhibition of the mutant and wild-type alleles are indicated (n=4, mean ± SDs). ( B ) Specific suppression of endogenous or oncogenic EGFR alleles. The si747/49_3D8 and si746/50_3D4 siRNAs were introduced into PC-3 and PC-9 human adenocarcinoma cells possessing the L747_E749del, A750P or E746_A750del mutations, respectively; endogenous EGFR mRNAs were examined using RT-PCR. Cells transfected with siControl (non-silencing siRNA) were studied as a control. M: DNA marker. ( C ) ASP-RNAi-mediated inhibition of PC-3 and PC-9 cell proliferation. Single siRNAs were transfected into PC-3 and PC-9 cells; subsequently, the cells were stained with Hoechst 33342 (blue) and propidium iodide (PI) (red) at the indicated time points, and examined using a fluorescent microscope. The numbers of total and dead cells were counted in four different 1-mm 2 areas. The data are averages of the four counts (± SDs; * P
Figure Legend Snippet: Oncogenic allele-specific RNAi. ( A ) Specific inhibition of mutant EGFR reporter alleles by ASP-RNAi. The effects of allele-specific siRNAs, si747/49_3D8 and si746/50_3D4, on expression of the target L747_E749del, A750P and E746_A750del EGFR mutant reporter alleles, respectively, and of the normal reporter alleles were examined using IC50 analysis (details in Methods). The IC50 values of the siRNAs for inhibition of the mutant and wild-type alleles are indicated (n=4, mean ± SDs). ( B ) Specific suppression of endogenous or oncogenic EGFR alleles. The si747/49_3D8 and si746/50_3D4 siRNAs were introduced into PC-3 and PC-9 human adenocarcinoma cells possessing the L747_E749del, A750P or E746_A750del mutations, respectively; endogenous EGFR mRNAs were examined using RT-PCR. Cells transfected with siControl (non-silencing siRNA) were studied as a control. M: DNA marker. ( C ) ASP-RNAi-mediated inhibition of PC-3 and PC-9 cell proliferation. Single siRNAs were transfected into PC-3 and PC-9 cells; subsequently, the cells were stained with Hoechst 33342 (blue) and propidium iodide (PI) (red) at the indicated time points, and examined using a fluorescent microscope. The numbers of total and dead cells were counted in four different 1-mm 2 areas. The data are averages of the four counts (± SDs; * P

Techniques Used: Inhibition, Mutagenesis, Expressing, Reverse Transcription Polymerase Chain Reaction, Transfection, Marker, Staining, Microscopy

18) Product Images from "Mechanistic studies of anticancer aptamer AS1411 reveal a novel role for nucleolin in regulating Rac1 activation"

Article Title: Mechanistic studies of anticancer aptamer AS1411 reveal a novel role for nucleolin in regulating Rac1 activation

Journal: Molecular Oncology

doi: 10.1016/j.molonc.2015.03.012

Effect of EGFR inhibition on AS1411‐induced signaling. DU145 cells were treated without added oligonucleotide (–), with 10 μM CRO control (C), or with 10 μM AS1411 (AS) for 48 h. Cells were washed
Figure Legend Snippet: Effect of EGFR inhibition on AS1411‐induced signaling. DU145 cells were treated without added oligonucleotide (–), with 10 μM CRO control (C), or with 10 μM AS1411 (AS) for 48 h. Cells were washed

Techniques Used: Inhibition

19) Product Images from "A Phosphotyrosine Proteomic Screen Identifies Multiple Tyrosine Kinase Signaling Pathways Aberrantly Activated in Malignant Mesothelioma"

Article Title: A Phosphotyrosine Proteomic Screen Identifies Multiple Tyrosine Kinase Signaling Pathways Aberrantly Activated in Malignant Mesothelioma

Journal: Genes & Cancer

doi: 10.1177/1947601910375273

Dual inhibition of concomitantly activated tyrosine kinases is more efficacious at inhibiting malignant mesothelioma (MM) cell viability and downstream effector signaling. ( A ) Meso 8 and Meso 43 cells were seeded on a 96-well plate and treated with increasing concentrations of AG1478 (EGFR inhibitor), MET Kinase Inhibitor, or PP2 (SFK inhibitor) for 72 hours, and then cell viability was determined by MTT assay. Western blot analysis was performed to evaluate a minimal concentration of each drug needed to inhibit EGFR, MET, and SFK phosphorylation, respectively (24 hours posttreatment). Total EGFR, MET, and Src, as well as actin, are included as loading controls. ( B ) Meso 8 and 43 cells were seeded on a 96-well plate and treated or not with AG1478 (E), MET Kinase Inhibitor (M), PP2 (P), AG1478 + MET Kinase Inhibitor (EM), MET Kinase Inhibitor + PP2 (MP), and AG1478 + PP2 (EP) for 72 hours, followed by assessment of cell viability by MTT assay. ( C ) Meso 8 and 43 cells were treated with the aforementioned drug combinations and were harvested 24 hours later for Western blot analysis. P-AKT, P-ERK1/2, and P-S6RP levels were evaluated to determine drug effects on AKT, MAPK, and mTOR activity, respectively. Total AKT, ERK1/2, and S6RP, as well as actin, are included as loading controls.
Figure Legend Snippet: Dual inhibition of concomitantly activated tyrosine kinases is more efficacious at inhibiting malignant mesothelioma (MM) cell viability and downstream effector signaling. ( A ) Meso 8 and Meso 43 cells were seeded on a 96-well plate and treated with increasing concentrations of AG1478 (EGFR inhibitor), MET Kinase Inhibitor, or PP2 (SFK inhibitor) for 72 hours, and then cell viability was determined by MTT assay. Western blot analysis was performed to evaluate a minimal concentration of each drug needed to inhibit EGFR, MET, and SFK phosphorylation, respectively (24 hours posttreatment). Total EGFR, MET, and Src, as well as actin, are included as loading controls. ( B ) Meso 8 and 43 cells were seeded on a 96-well plate and treated or not with AG1478 (E), MET Kinase Inhibitor (M), PP2 (P), AG1478 + MET Kinase Inhibitor (EM), MET Kinase Inhibitor + PP2 (MP), and AG1478 + PP2 (EP) for 72 hours, followed by assessment of cell viability by MTT assay. ( C ) Meso 8 and 43 cells were treated with the aforementioned drug combinations and were harvested 24 hours later for Western blot analysis. P-AKT, P-ERK1/2, and P-S6RP levels were evaluated to determine drug effects on AKT, MAPK, and mTOR activity, respectively. Total AKT, ERK1/2, and S6RP, as well as actin, are included as loading controls.

Techniques Used: Inhibition, MTT Assay, Western Blot, Concentration Assay, Activity Assay

20) Product Images from "Luteolin inhibits Musashi1 binding to RNA and disrupts cancer phenotypes in glioblastoma cells"

Article Title: Luteolin inhibits Musashi1 binding to RNA and disrupts cancer phenotypes in glioblastoma cells

Journal: RNA Biology

doi: 10.1080/15476286.2018.1539607

Luteolin impacts the expression of Musashi1 target genes. a) Western blot showing the impact of luteolin treatment on the expression of Msi1 target genes (PDGFRα, IGF-IR, EGFR, CCND1 and CDK6) 48 hours post-treatment. Msi1 functions as a positive regulator of these transcripts by increasing their translation or stability. Tubulin was used as loading control. b) We used a luciferase reporter containing the 3’UTR of PDGFRα to corroborate that luteolin treatment disrupts Msi1 regulatory functions. Msi1 is a positive regulator of PDGFRα translation. HT1080 cells were co-transfected with plasmids expressing either GST or Msi1. Msi1 expression affected the expression of the reporter, resulting increased luciferase activity. The effect was largely diminished when luteolin was added after transfection. Experiments were done in triplicate. A representative western is shown. Statistical significance was calculated by multiple t test. All data are shown as means ± s.d. (*PÂÂ
Figure Legend Snippet: Luteolin impacts the expression of Musashi1 target genes. a) Western blot showing the impact of luteolin treatment on the expression of Msi1 target genes (PDGFRα, IGF-IR, EGFR, CCND1 and CDK6) 48 hours post-treatment. Msi1 functions as a positive regulator of these transcripts by increasing their translation or stability. Tubulin was used as loading control. b) We used a luciferase reporter containing the 3’UTR of PDGFRα to corroborate that luteolin treatment disrupts Msi1 regulatory functions. Msi1 is a positive regulator of PDGFRα translation. HT1080 cells were co-transfected with plasmids expressing either GST or Msi1. Msi1 expression affected the expression of the reporter, resulting increased luciferase activity. The effect was largely diminished when luteolin was added after transfection. Experiments were done in triplicate. A representative western is shown. Statistical significance was calculated by multiple t test. All data are shown as means ± s.d. (*PÂÂ

Techniques Used: Expressing, Western Blot, Luciferase, Transfection, Activity Assay

21) Product Images from "MUC1 Contributes to BPDE-Induced Human Bronchial Epithelial Cell Transformation through Facilitating EGFR Activation"

Article Title: MUC1 Contributes to BPDE-Induced Human Bronchial Epithelial Cell Transformation through Facilitating EGFR Activation

Journal: PLoS ONE

doi: 10.1371/journal.pone.0033846

Chronic BPDE exposure activates Akt and ERK through EGFR in human bronchial epithelial cells. A , Induction of MUC1 expression and EGFR-, Akt- and ERK-activation in HBEC-2 cells by BPDE. Specifically, HBEC-2 cells were treated with the vehicle DMSO or BPDE (0.1 µM) for the indicated weeks. Western blot was the same as in A . β-Actin was detected as a loading control. B , Increased MUC1 expression and EGFR-, Akt- and ERK-activation in transformed HBEC-2 cells by BPDE. HBEC-2 cells were treated with BPDE (0.1 µM) for 12 wk and then seeded in soft agar. Colonies were grown up for 3 wk in transformed cells (TRANS). Wild-type (WT, exposed to sham) HBEC-2 cells were as a negative control. Expression of MUC1 and activation of Akt and ERK were detected by Western blot in both WT and the transfected cells. β-Actin was detected as a loading control.
Figure Legend Snippet: Chronic BPDE exposure activates Akt and ERK through EGFR in human bronchial epithelial cells. A , Induction of MUC1 expression and EGFR-, Akt- and ERK-activation in HBEC-2 cells by BPDE. Specifically, HBEC-2 cells were treated with the vehicle DMSO or BPDE (0.1 µM) for the indicated weeks. Western blot was the same as in A . β-Actin was detected as a loading control. B , Increased MUC1 expression and EGFR-, Akt- and ERK-activation in transformed HBEC-2 cells by BPDE. HBEC-2 cells were treated with BPDE (0.1 µM) for 12 wk and then seeded in soft agar. Colonies were grown up for 3 wk in transformed cells (TRANS). Wild-type (WT, exposed to sham) HBEC-2 cells were as a negative control. Expression of MUC1 and activation of Akt and ERK were detected by Western blot in both WT and the transfected cells. β-Actin was detected as a loading control.

Techniques Used: Expressing, Activation Assay, Western Blot, Transformation Assay, Negative Control, Transfection

Suppression of EGFR, Akt and ERK activation inhibits BPDE-induced cell transformation. Graphical and quantitative representation of colony formation in soft agar of BEAS-2B cells exposed to BPDE (0.1 µM) and/or the indicated inhibitors (EGFRin, LY, and U0126) every two days for 1 week. Bars show the averages of colony numbers of 6 randomly selected fields. Data shown are mean ± S.D; ** P
Figure Legend Snippet: Suppression of EGFR, Akt and ERK activation inhibits BPDE-induced cell transformation. Graphical and quantitative representation of colony formation in soft agar of BEAS-2B cells exposed to BPDE (0.1 µM) and/or the indicated inhibitors (EGFRin, LY, and U0126) every two days for 1 week. Bars show the averages of colony numbers of 6 randomly selected fields. Data shown are mean ± S.D; ** P

Techniques Used: Activation Assay, Transformation Assay

Blocking EGFR, Akt and ERK activation potentiates BPDE-induced cytotoxicity. BEAS-2B cells were pretreated with the indicated inhibitors (LY (10 µM) for Akt, EGFRin (6 µM) for EGFR, U0126 (5 µM) for ERK, and SP (10 µM) for JNK) for 30 min followed by exposure to BPDE (0.2 µM) for 48 hr. Cell viability was detected by LDH release and MTT assays. Data shown are mean ± S.D; ** P
Figure Legend Snippet: Blocking EGFR, Akt and ERK activation potentiates BPDE-induced cytotoxicity. BEAS-2B cells were pretreated with the indicated inhibitors (LY (10 µM) for Akt, EGFRin (6 µM) for EGFR, U0126 (5 µM) for ERK, and SP (10 µM) for JNK) for 30 min followed by exposure to BPDE (0.2 µM) for 48 hr. Cell viability was detected by LDH release and MTT assays. Data shown are mean ± S.D; ** P

Techniques Used: Blocking Assay, Activation Assay, MTT Assay

Transient BPDE exposure activates Akt and ERK through EGFR in human bronchial epithelial cells. A , Acute BPDE exposure induces activation of EGFR, Akt, and ERK in BEAS-2B cells. BEAS-2B cells were treated with the indicated concentrations of BPDE for 2 hr. Activation of EGFR, Akt and ERK were detected by Western blot with antibodies recognizing each phosphorylated protein (phosphorylation sites are indicated). β-Actin was detected as an input control. B , Suppression of EGFR attenuates the activation of Akt and ERK in BEAS-2B cells. The expression and activity of EGFR in BEAS-2B cells were suppressed with either EGFR inhibitor III or EGFR siRNA. The cells were then exposed to BPDE (0.4 µM) for 2 hr. The indicated proteins were detected by Western blot. β-Actin was detected as a loading control.
Figure Legend Snippet: Transient BPDE exposure activates Akt and ERK through EGFR in human bronchial epithelial cells. A , Acute BPDE exposure induces activation of EGFR, Akt, and ERK in BEAS-2B cells. BEAS-2B cells were treated with the indicated concentrations of BPDE for 2 hr. Activation of EGFR, Akt and ERK were detected by Western blot with antibodies recognizing each phosphorylated protein (phosphorylation sites are indicated). β-Actin was detected as an input control. B , Suppression of EGFR attenuates the activation of Akt and ERK in BEAS-2B cells. The expression and activity of EGFR in BEAS-2B cells were suppressed with either EGFR inhibitor III or EGFR siRNA. The cells were then exposed to BPDE (0.4 µM) for 2 hr. The indicated proteins were detected by Western blot. β-Actin was detected as a loading control.

Techniques Used: Activation Assay, Western Blot, Expressing, Activity Assay

MUC1 stabilizes EGFR, contributes to BPDE-induced EGFR, Akt and ERK activation in BEAS-2B cells and protects cells from BPDE-induced cytotoxicity. A , MUC1 is required for BPDE-induced EGFR, Akt and ERK activation. Cells stably transfected with MUC1 shRNA or negative control shRNA were treated with BPDE for the indicated time periods. Activation of each protein was detected with antibodies against the phosphorylated form of the proteins. The phosphorylation sites of each protein are indicated. Total EGFR, Akt and ERK were also detected. β-Actin was detected as an input control. B , Reduced EGFR expression in MUC1 Knockdown cells. Upper left , equal amounts of total RNA from the indicated cells were detected for EGFR mRNA expression. β-Actin was detected as an input control. Upper right , Cells stably transfected with MUC1 shRNA or negative control shRNA were treated with cycloheximide (CHX, 10 µM) for the indicated time periods. EGFR protein was detected by Western blot. β-Actin was detected as an input control. Lower right , quantification of the results of Upper right . The intensity of the individual bands was quantified by densitometry (NIH Image 1.62) and normalized to the corresponding input control (β-actin) bands. EGFR expression changes were calculated with the control taken as 100%. C , BPDE-induced cytotoxicity is increased in MUC1 knockdown cells. BEAS-2B WT and MUC1 knockdown cells were treated with the indicated concentrations of BPDE for 48 hr. Cell viability was detected by LDH release and MTT assays. Data shown are mean ± S.D; **P
Figure Legend Snippet: MUC1 stabilizes EGFR, contributes to BPDE-induced EGFR, Akt and ERK activation in BEAS-2B cells and protects cells from BPDE-induced cytotoxicity. A , MUC1 is required for BPDE-induced EGFR, Akt and ERK activation. Cells stably transfected with MUC1 shRNA or negative control shRNA were treated with BPDE for the indicated time periods. Activation of each protein was detected with antibodies against the phosphorylated form of the proteins. The phosphorylation sites of each protein are indicated. Total EGFR, Akt and ERK were also detected. β-Actin was detected as an input control. B , Reduced EGFR expression in MUC1 Knockdown cells. Upper left , equal amounts of total RNA from the indicated cells were detected for EGFR mRNA expression. β-Actin was detected as an input control. Upper right , Cells stably transfected with MUC1 shRNA or negative control shRNA were treated with cycloheximide (CHX, 10 µM) for the indicated time periods. EGFR protein was detected by Western blot. β-Actin was detected as an input control. Lower right , quantification of the results of Upper right . The intensity of the individual bands was quantified by densitometry (NIH Image 1.62) and normalized to the corresponding input control (β-actin) bands. EGFR expression changes were calculated with the control taken as 100%. C , BPDE-induced cytotoxicity is increased in MUC1 knockdown cells. BEAS-2B WT and MUC1 knockdown cells were treated with the indicated concentrations of BPDE for 48 hr. Cell viability was detected by LDH release and MTT assays. Data shown are mean ± S.D; **P

Techniques Used: Activation Assay, Stable Transfection, Transfection, shRNA, Negative Control, Expressing, Western Blot, MTT Assay

A model of MUC1-mediated EGFR activation and HBEC transformation. CS carcinogens such as BPDE trigger MUC1 expression in bronchial epithelial cells, facilitating EGFR-mediated cell survival signaling via Akt and ERK activation. Akt and ERK protect cells against DNA damage-mediated apoptosis to promote cell transformation, facilitating lung carcinogenesis.
Figure Legend Snippet: A model of MUC1-mediated EGFR activation and HBEC transformation. CS carcinogens such as BPDE trigger MUC1 expression in bronchial epithelial cells, facilitating EGFR-mediated cell survival signaling via Akt and ERK activation. Akt and ERK protect cells against DNA damage-mediated apoptosis to promote cell transformation, facilitating lung carcinogenesis.

Techniques Used: Activation Assay, Transformation Assay, Expressing

22) Product Images from "Temporal regulation of EGF signaling networks by the scaffold protein Shc1"

Article Title: Temporal regulation of EGF signaling networks by the scaffold protein Shc1

Journal: Nature

doi: 10.1038/nature12308

Dynamic phosphorylation of Shc1 and interacting proteins a, Temporal profiles of individual Shc1 phosphorylation sites following EGF stimulation. dt-Shc1 was affinity purified from EGF-stimulated fibroblasts at various time points. Relative abundance of dt-Shc1 phosphopeptides was quantified by sMRM and plotted using a quasi logarithm time scale to expand the early phase of phosphorylation. b, Temporal profiles of all analyzed phosphorylation sites in the EGF-induced Shc1 complex. The size of each dot is proportional to the relative abundance of the corresponding phosphopeptide. c, Differential inhibition of Shc1 phosphorylation by kinase inhibitors as quantified by sMRM. d, In vitro kinase/sMRM analysis. Affinity purified dt-Shc1 was incubated with recombinant kinases in vitro . Phosphorylation of dt-Shc1 sites was quantified by sMRM. e , Activation kinetics of Akt and Erk1 were measured by quantitative immunoblotting, and overlaid with the phosphorylation kinetics of Shc1 S29 and T214 from panel a , respectively. Inhibitors used: EGFR: AG1478; PI3K: LY294002; Mek: PD98059. Results are representative of three independent experiments. Error bars are s. d. from all transitions for a given protein/peptide from all technical repeats.
Figure Legend Snippet: Dynamic phosphorylation of Shc1 and interacting proteins a, Temporal profiles of individual Shc1 phosphorylation sites following EGF stimulation. dt-Shc1 was affinity purified from EGF-stimulated fibroblasts at various time points. Relative abundance of dt-Shc1 phosphopeptides was quantified by sMRM and plotted using a quasi logarithm time scale to expand the early phase of phosphorylation. b, Temporal profiles of all analyzed phosphorylation sites in the EGF-induced Shc1 complex. The size of each dot is proportional to the relative abundance of the corresponding phosphopeptide. c, Differential inhibition of Shc1 phosphorylation by kinase inhibitors as quantified by sMRM. d, In vitro kinase/sMRM analysis. Affinity purified dt-Shc1 was incubated with recombinant kinases in vitro . Phosphorylation of dt-Shc1 sites was quantified by sMRM. e , Activation kinetics of Akt and Erk1 were measured by quantitative immunoblotting, and overlaid with the phosphorylation kinetics of Shc1 S29 and T214 from panel a , respectively. Inhibitors used: EGFR: AG1478; PI3K: LY294002; Mek: PD98059. Results are representative of three independent experiments. Error bars are s. d. from all transitions for a given protein/peptide from all technical repeats.

Techniques Used: Affinity Purification, Inhibition, In Vitro, Incubation, Recombinant, Activation Assay

23) Product Images from "SR48692 inhibits non-small cell lung cancer proliferation in an EGF receptor-dependent manner"

Article Title: SR48692 inhibits non-small cell lung cancer proliferation in an EGF receptor-dependent manner

Journal: Life sciences

doi: 10.1016/j.lfs.2014.01.072

Inhibition of EGFR and ERK tyrosine phosphorylation by SR48692. (A) The ability of NTS to increase EGFR or ERK tyrosine phosphorylation was investigated as a function of SR48692 (SR) concentration using NCI-H1299 cells. The P-EGFR and total EGFR is shown.
Figure Legend Snippet: Inhibition of EGFR and ERK tyrosine phosphorylation by SR48692. (A) The ability of NTS to increase EGFR or ERK tyrosine phosphorylation was investigated as a function of SR48692 (SR) concentration using NCI-H1299 cells. The P-EGFR and total EGFR is shown.

Techniques Used: Inhibition, Concentration Assay

Inhibition of EGFR and ERK tyrosine phosphorylation by gefitinib. (A) The ability of NTS to caused EGFR tyrosine phosphorylation was investigated as a function of gefitinib concentration using NCI-H1299 cells. The P-EGFR and total EGFR is shown. (B) The
Figure Legend Snippet: Inhibition of EGFR and ERK tyrosine phosphorylation by gefitinib. (A) The ability of NTS to caused EGFR tyrosine phosphorylation was investigated as a function of gefitinib concentration using NCI-H1299 cells. The P-EGFR and total EGFR is shown. (B) The

Techniques Used: Inhibition, Concentration Assay

24) Product Images from "Role for engagement of β‐arrestin2 by the transactivated EGFR in agonist‐specific regulation of δ receptor activation of ERK1/2) Role for engagement of β‐arrestin2 by the transactivated EGFR in agonist‐specific regulation of δ receptor activation of ERK1/2"

Article Title: Role for engagement of β‐arrestin2 by the transactivated EGFR in agonist‐specific regulation of δ receptor activation of ERK1/2) Role for engagement of β‐arrestin2 by the transactivated EGFR in agonist‐specific regulation of δ receptor activation of ERK1/2

Journal: British Journal of Pharmacology

doi: 10.1111/bph.13254

Schematic representation of δ receptor‐mediated ERK1/2 activation via the PKCδ/EGFR/β‐arrestin2 signalling pathway in response to specific agonist stimulation. DPDPE binding to δ receptors results in the
Figure Legend Snippet: Schematic representation of δ receptor‐mediated ERK1/2 activation via the PKCδ/EGFR/β‐arrestin2 signalling pathway in response to specific agonist stimulation. DPDPE binding to δ receptors results in the

Techniques Used: Activation Assay, Binding Assay

PKCδ acts upstream of metalloproteinase activation and is required for the release of the EGFR‐activating factor. (A) Serum‐starved δ receptor‐null receiving cells previously treated or not for 30 min with
Figure Legend Snippet: PKCδ acts upstream of metalloproteinase activation and is required for the release of the EGFR‐activating factor. (A) Serum‐starved δ receptor‐null receiving cells previously treated or not for 30 min with

Techniques Used: Activation Assay

β‐Arrestin2 is required for DPDPE‐stimulated ERK1/2 activation and acts downstream of the PKCδ‐mediated activation of metalloproteinase and transactivation of EGFR in HEK293 cells stably expressing the S363A δ
Figure Legend Snippet: β‐Arrestin2 is required for DPDPE‐stimulated ERK1/2 activation and acts downstream of the PKCδ‐mediated activation of metalloproteinase and transactivation of EGFR in HEK293 cells stably expressing the S363A δ

Techniques Used: Activation Assay, Stable Transfection, Expressing

δ Receptor‐mediated ERK1/2 activation stimulated by TIPP or morphine but not by DPDPE involves the transactivation of EGFR in cultured cells and native tissues. (A, B) Serum‐starved HEK293 δ receptor cells or NG108‐15
Figure Legend Snippet: δ Receptor‐mediated ERK1/2 activation stimulated by TIPP or morphine but not by DPDPE involves the transactivation of EGFR in cultured cells and native tissues. (A, B) Serum‐starved HEK293 δ receptor cells or NG108‐15

Techniques Used: Activation Assay, Cell Culture

β‐Arrestin2 is required for δ receptor‐mediated ERK1/2 activation stimulated by morphine and TIPP but not by DPDPE and acts downstream of EGFR transactivation. (A) HEK293 δ receptor cells were transfected with siRNAs
Figure Legend Snippet: β‐Arrestin2 is required for δ receptor‐mediated ERK1/2 activation stimulated by morphine and TIPP but not by DPDPE and acts downstream of EGFR transactivation. (A) HEK293 δ receptor cells were transfected with siRNAs

Techniques Used: Activation Assay, Transfection

DPDPE can activate ERK1/2 by employing β‐arrestin2 via PKCδ‐mediated transactivation of EGFR in HEK293 cells expressing the S363A mutant δ receptor. (A) Cells stably expressing wild‐type δ receptors
Figure Legend Snippet: DPDPE can activate ERK1/2 by employing β‐arrestin2 via PKCδ‐mediated transactivation of EGFR in HEK293 cells expressing the S363A mutant δ receptor. (A) Cells stably expressing wild‐type δ receptors

Techniques Used: Expressing, Mutagenesis, Stable Transfection

PKCδ activity is required for δ receptor‐mediated ERK1/2 activation via transactivation of EGFR in response to TIPP or morphine but not DPDPE stimulation. (A) Serum‐starved HEK293 δ receptor cells were pretreated
Figure Legend Snippet: PKCδ activity is required for δ receptor‐mediated ERK1/2 activation via transactivation of EGFR in response to TIPP or morphine but not DPDPE stimulation. (A) Serum‐starved HEK293 δ receptor cells were pretreated

Techniques Used: Activity Assay, Activation Assay

25) Product Images from "Mineralocorticoid receptor interaction with SP1 generates a new response element for pathophysiologically relevant gene expression"

Article Title: Mineralocorticoid receptor interaction with SP1 generates a new response element for pathophysiologically relevant gene expression

Journal: Nucleic Acids Research

doi: 10.1093/nar/gkt581

Relevance of SP1 for aldosterone-induced EGFR expression in primary culture. ( A ) HAoSMC were incubated for 24 h with either vehicle, 10 nM aldosterone, 1 µM WP631 or 10 nM aldosterone and 1 µM WP631 and EGFR protein expression was quantified by western blot and densitometric analysis ( n = 5, data represented as mean ± SEM, * P ≤ 0.05). ( B ) Primary rat aortic smooth muscle cells (A7r5) were incubated for 24 h with either vehicle, 10 nM aldosterone, 1 µM WP631 or 10 nM aldosterone and 1 µM WP631 and EGFR protein expression was quantified by western blot and densitometric analysis ( n = 3, data represented as mean ± SEM, * P ≤ 0.05).
Figure Legend Snippet: Relevance of SP1 for aldosterone-induced EGFR expression in primary culture. ( A ) HAoSMC were incubated for 24 h with either vehicle, 10 nM aldosterone, 1 µM WP631 or 10 nM aldosterone and 1 µM WP631 and EGFR protein expression was quantified by western blot and densitometric analysis ( n = 5, data represented as mean ± SEM, * P ≤ 0.05). ( B ) Primary rat aortic smooth muscle cells (A7r5) were incubated for 24 h with either vehicle, 10 nM aldosterone, 1 µM WP631 or 10 nM aldosterone and 1 µM WP631 and EGFR protein expression was quantified by western blot and densitometric analysis ( n = 3, data represented as mean ± SEM, * P ≤ 0.05).

Techniques Used: Expressing, Incubation, Western Blot

26) Product Images from "Epidermal Growth Factor Receptor Activation in Glioblastoma through Novel Missense Mutations in the Extracellular DomainA Genome-Wide Screen for Promoter Methylation in Lung Cancer Identifies Novel Methylation Markers for Multiple Malignancies "

Article Title: Epidermal Growth Factor Receptor Activation in Glioblastoma through Novel Missense Mutations in the Extracellular DomainA Genome-Wide Screen for Promoter Methylation in Lung Cancer Identifies Novel Methylation Markers for Multiple Malignancies

Journal: PLoS Medicine

doi: 10.1371/journal.pmed.0030485

Basal Activation and Ligand Response of EGFR Ectodomain Mutants (A) Increased EGFR tyrosine phosphorylation of A289V-EGFR. 293T cells were transiently transfected with green fluorescent protein (GFP; control), wild-type EGFR , or A289V- EGFR . At 24 h after transfection, 24 cells were serum starved for 12 h and then lysed. Shown are immunoblots of immunoprecipitated EGFR (left blots) and whole cell lysates (right blots). (B) Increased basal activity of EGFR missense mutants in human astrocytes. Immortalized human astrocytes were stably infected with wild-type EGFR or the indicated EGFR missense mutants. Shown are total phosphotyrosine (PY), Y1068-EGFR, total EGFR, and PI3K p85 (loading control) immunoblots of whole cell lysates from cells following 12 h of serum starvation. The solid arrow at the PY position represents tyrosine-phosphorylated EGFR, and the interrupted arrows indicated other differentially tyrosine-phosphorylated proteins. The inset shows an anti-EGFR immunoblot of parental astrocytes (far-left lane) and stable astrocyte subclones (designated in remaining five lanes) growing in full serum. (C) Basal receptor phosphorylation and EGF-responsiveness of wild-type EGFR and four different EGFR ectodomain mutants stably expressed in Ba/F3 murine hematopoietic cells. Shown are immunoblots of stable Ba/F3 subclones after 12 h of serum starvation (− EGF) and 15 min following EGF-induction (0.5 or 5 ng/ml EGF).
Figure Legend Snippet: Basal Activation and Ligand Response of EGFR Ectodomain Mutants (A) Increased EGFR tyrosine phosphorylation of A289V-EGFR. 293T cells were transiently transfected with green fluorescent protein (GFP; control), wild-type EGFR , or A289V- EGFR . At 24 h after transfection, 24 cells were serum starved for 12 h and then lysed. Shown are immunoblots of immunoprecipitated EGFR (left blots) and whole cell lysates (right blots). (B) Increased basal activity of EGFR missense mutants in human astrocytes. Immortalized human astrocytes were stably infected with wild-type EGFR or the indicated EGFR missense mutants. Shown are total phosphotyrosine (PY), Y1068-EGFR, total EGFR, and PI3K p85 (loading control) immunoblots of whole cell lysates from cells following 12 h of serum starvation. The solid arrow at the PY position represents tyrosine-phosphorylated EGFR, and the interrupted arrows indicated other differentially tyrosine-phosphorylated proteins. The inset shows an anti-EGFR immunoblot of parental astrocytes (far-left lane) and stable astrocyte subclones (designated in remaining five lanes) growing in full serum. (C) Basal receptor phosphorylation and EGF-responsiveness of wild-type EGFR and four different EGFR ectodomain mutants stably expressed in Ba/F3 murine hematopoietic cells. Shown are immunoblots of stable Ba/F3 subclones after 12 h of serum starvation (− EGF) and 15 min following EGF-induction (0.5 or 5 ng/ml EGF).

Techniques Used: Activation Assay, Transfection, Western Blot, Immunoprecipitation, Activity Assay, Stable Transfection, Infection

27) Product Images from "EGFR is Essential for TLR3 Signaling"

Article Title: EGFR is Essential for TLR3 Signaling

Journal: Science signaling

doi: 10.1126/scisignal.2002581

EGFR is essential for TLR3 signaling
Figure Legend Snippet: EGFR is essential for TLR3 signaling

Techniques Used:

EGFR is required for activation of IRF-3 and recruitment of TRIF by TLR3
Figure Legend Snippet: EGFR is required for activation of IRF-3 and recruitment of TRIF by TLR3

Techniques Used: Activation Assay

EGFR interaction with TLR3 is independent of TLR3 tyrosine residues and TRIF
Figure Legend Snippet: EGFR interaction with TLR3 is independent of TLR3 tyrosine residues and TRIF

Techniques Used:

Src interaction with TLR3 is independent of TRIF and TRAF3, but dependent on EGFR
Figure Legend Snippet: Src interaction with TLR3 is independent of TRIF and TRAF3, but dependent on EGFR

Techniques Used:

Src and EGFR are required for phosphorylation of Tyr 759 and Tyr 858 in TLR3
Figure Legend Snippet: Src and EGFR are required for phosphorylation of Tyr 759 and Tyr 858 in TLR3

Techniques Used:

The TIR domain of TLR3 is not required for its interaction with Src and EGFR
Figure Legend Snippet: The TIR domain of TLR3 is not required for its interaction with Src and EGFR

Techniques Used:

28) Product Images from "PEAK1, acting as a tumor promoter in colorectal cancer, is regulated by the EGFR/KRas signaling axis and miR-181d"

Article Title: PEAK1, acting as a tumor promoter in colorectal cancer, is regulated by the EGFR/KRas signaling axis and miR-181d

Journal: Cell Death & Disease

doi: 10.1038/s41419-018-0320-8

EGFR signaling increases the expression of PEAK1. a The correlation between EGFR and PEAK1 expression was evaluated by Spearman’s method. b Western blot analyses of EGFR, PEAK1 and Erk in total extracts from CRC cells treated with EGF for 1 h. c CRC cells were treated with EGF, followed by siPEAK1 transfection, and then western blot was performed to analyze the Erk levels. These experiments were repeated three times
Figure Legend Snippet: EGFR signaling increases the expression of PEAK1. a The correlation between EGFR and PEAK1 expression was evaluated by Spearman’s method. b Western blot analyses of EGFR, PEAK1 and Erk in total extracts from CRC cells treated with EGF for 1 h. c CRC cells were treated with EGF, followed by siPEAK1 transfection, and then western blot was performed to analyze the Erk levels. These experiments were repeated three times

Techniques Used: Expressing, Western Blot, Transfection

29) Product Images from "Icotinib hydrochloride enhances chemo- and radiosensitivity by inhibiting EGFR signaling and attenuating RAD51 expression and function in Hela S3 cells"

Article Title: Icotinib hydrochloride enhances chemo- and radiosensitivity by inhibiting EGFR signaling and attenuating RAD51 expression and function in Hela S3 cells

Journal: OncoTargets and therapy

doi: 10.2147/OTT.S152613

Icotinib hydrochloride inhibits EGFR signaling pathway in Hela S3 cells. Notes: Total EGFR, p-EGFR, total AKT, p-AKT, total ERK and p-ERK were detected using Western blot assays in Hela S3 cells 2 hours after combination treatment of radiation ( A ) or cisplatin ( B ) with IH (top). Histograms indicate the relative protein expression levels in the cells by grayscale analysis (bottom). Error bars, SD. * P
Figure Legend Snippet: Icotinib hydrochloride inhibits EGFR signaling pathway in Hela S3 cells. Notes: Total EGFR, p-EGFR, total AKT, p-AKT, total ERK and p-ERK were detected using Western blot assays in Hela S3 cells 2 hours after combination treatment of radiation ( A ) or cisplatin ( B ) with IH (top). Histograms indicate the relative protein expression levels in the cells by grayscale analysis (bottom). Error bars, SD. * P

Techniques Used: Western Blot, Expressing

30) Product Images from "KRAS mutant allele-specific expression knockdown in pancreatic cancer model with systemically delivered bi-shRNA KRAS lipoplex"

Article Title: KRAS mutant allele-specific expression knockdown in pancreatic cancer model with systemically delivered bi-shRNA KRAS lipoplex

Journal: PLoS ONE

doi: 10.1371/journal.pone.0193644

Molecular analysis of in vivo tumor samples. A. Electropherogram analyze % of mu and wt KRAS transcripts in in vivo treated tumor samples. Tumors were removed from animals after four weeks of various treatments, proportions of mu and wt KRAS transcripts were analyzed by RFLP and assayed by Experion. Sample designation is the same as indicated for Fig 4B . % mu and wt KRAS transcripts show at the bottom of the figure was determined by Experion software. B. Western transfer shows protein expression in various tumor samples. Numbers on each sample indicate treatment groups as presented in Fig 4 . Two independent isolated tumors are analyzed for each treatment group. Panel a shows RAS protein in various treated tumor samples normalized against GAPDH. Panel b shows p-EGFR at position Y1068 quantitatively normalized against total EGFR protein. Panel c shows total EGFR protein for various treatment groups. C. Bar graphs summarize fold intensity difference from various groups of in vivo samples. Sample groupings are the same as shown on Fig 4 . Panel a is for p-EGFR at Y1045 normalized to total EGFR protein. Panel b is for p-EGFR at Y1068 normalized to total EGFR protein. Panel c is for p-EGFR at Y1125 normalized to total EGFR protein. Panel d is total EGFR protein normalized to GAPDH. For each sample n = 3. Bar graphs shown are data obtained from approximately half of tumor of three independent animals. Standard deviation bar represents measurements of tumor from three animals. With one tailed, equal variances, student T-test, the following samples show statistical significant ρ-value ≤ 0.05: Panel a between samples 1 and 6, Panel c between samples 1 and 5, Panel d between samples 1 and 4, 2 and 4, 2 and 6.
Figure Legend Snippet: Molecular analysis of in vivo tumor samples. A. Electropherogram analyze % of mu and wt KRAS transcripts in in vivo treated tumor samples. Tumors were removed from animals after four weeks of various treatments, proportions of mu and wt KRAS transcripts were analyzed by RFLP and assayed by Experion. Sample designation is the same as indicated for Fig 4B . % mu and wt KRAS transcripts show at the bottom of the figure was determined by Experion software. B. Western transfer shows protein expression in various tumor samples. Numbers on each sample indicate treatment groups as presented in Fig 4 . Two independent isolated tumors are analyzed for each treatment group. Panel a shows RAS protein in various treated tumor samples normalized against GAPDH. Panel b shows p-EGFR at position Y1068 quantitatively normalized against total EGFR protein. Panel c shows total EGFR protein for various treatment groups. C. Bar graphs summarize fold intensity difference from various groups of in vivo samples. Sample groupings are the same as shown on Fig 4 . Panel a is for p-EGFR at Y1045 normalized to total EGFR protein. Panel b is for p-EGFR at Y1068 normalized to total EGFR protein. Panel c is for p-EGFR at Y1125 normalized to total EGFR protein. Panel d is total EGFR protein normalized to GAPDH. For each sample n = 3. Bar graphs shown are data obtained from approximately half of tumor of three independent animals. Standard deviation bar represents measurements of tumor from three animals. With one tailed, equal variances, student T-test, the following samples show statistical significant ρ-value ≤ 0.05: Panel a between samples 1 and 6, Panel c between samples 1 and 5, Panel d between samples 1 and 4, 2 and 4, 2 and 6.

Techniques Used: In Vivo, Software, Western Blot, Expressing, Isolation, Standard Deviation, One-tailed Test

31) Product Images from "Intracellular activation of EGFR by fatty acid synthase dependent palmitoylation"

Article Title: Intracellular activation of EGFR by fatty acid synthase dependent palmitoylation

Journal: Oncotarget

doi:

Inhibition of de novo fatty acid synthesis or palmitoylation alters EGFR cellular distribution and reduces total EGFR levels a. Representative immuno fluorescent images of PC3 cells. PC3 cells were treated with DMSO or cerulenin at 5 ug/ml for 24 hours and stained for EGFR (green), lysosomes (red) and nucleus (DAPI, blue). Images were taken using Olympus confocal microscope using 60x objective. Scale bar is 30um. b. Western blot analysis of protein samples for EGFR and Actin isolated from PC3 cells and A549 cells treated with FASN inhibitor, cerulenin at 5ug/ml or 2-BP (6 uM) for 24 hours.
Figure Legend Snippet: Inhibition of de novo fatty acid synthesis or palmitoylation alters EGFR cellular distribution and reduces total EGFR levels a. Representative immuno fluorescent images of PC3 cells. PC3 cells were treated with DMSO or cerulenin at 5 ug/ml for 24 hours and stained for EGFR (green), lysosomes (red) and nucleus (DAPI, blue). Images were taken using Olympus confocal microscope using 60x objective. Scale bar is 30um. b. Western blot analysis of protein samples for EGFR and Actin isolated from PC3 cells and A549 cells treated with FASN inhibitor, cerulenin at 5ug/ml or 2-BP (6 uM) for 24 hours.

Techniques Used: Inhibition, Staining, Microscopy, Western Blot, Isolation

PAT 1, 2 and 21 increases EGFR activation and palmitoylation a. Screening of PAT enzymes for EGFR activation. A549 cells were transfected with individual PAT (HA-tagged) constructs for 24 hours followed by 12 hours of serum starvation. Isolated protein samples were tested for EGFR activation using pEGFR antibody. b. Western blot analysis of protein samples for pEGFR, EGFR, pAkt, Akt, pErk 1/2, Erk 1/2 and Actin isolated from A549 cells transfected with PAT plasmids 1, 2, 21 or vector alone for 24 hours followed by serum starvation for 12 hours. c. Western blot analysis of immunoprecipitated samples for HA and EGFR antibodies. HEK 293 cells were transfected with EGFR-flag alone or in combination with indicated PATs as shown in the Figure 3c and PAT enzymes were immunoprecipitated with HA antibody. d. Western blotting analysis of palmitoylated EGFR from PC3 cells transfected with vector alone or PATs.
Figure Legend Snippet: PAT 1, 2 and 21 increases EGFR activation and palmitoylation a. Screening of PAT enzymes for EGFR activation. A549 cells were transfected with individual PAT (HA-tagged) constructs for 24 hours followed by 12 hours of serum starvation. Isolated protein samples were tested for EGFR activation using pEGFR antibody. b. Western blot analysis of protein samples for pEGFR, EGFR, pAkt, Akt, pErk 1/2, Erk 1/2 and Actin isolated from A549 cells transfected with PAT plasmids 1, 2, 21 or vector alone for 24 hours followed by serum starvation for 12 hours. c. Western blot analysis of immunoprecipitated samples for HA and EGFR antibodies. HEK 293 cells were transfected with EGFR-flag alone or in combination with indicated PATs as shown in the Figure 3c and PAT enzymes were immunoprecipitated with HA antibody. d. Western blotting analysis of palmitoylated EGFR from PC3 cells transfected with vector alone or PATs.

Techniques Used: Activation Assay, Transfection, Construct, Isolation, Western Blot, Plasmid Preparation, Immunoprecipitation

Constitutive activation of EGFR sustains cell proliferation in the absence of ligands a. Serum starved PC3, DU145, A549 and HT-29 cells were tested for EGFR phosphorylation. b. PC3 and A549 cells grown in the absence of serum/ligands were tested for EGFR dimerization (crosslinked by DMP) and phosphorylation. Serum starved A549 and PC3 were treated with EGF +/− C225 at 2.5 ug/ml c. or AEE788 at 2.5 uM d. for 15 minutes and measured for EGFR phosphorylation, EGFR and Actin using Western blot. e. Schematic diagram of WT and extra cellular domain deleted EGFR (ΔECD-EGFR). f. Western blot analysis of protein samples for pEGFR and EGFR (Flag) isolated from HEK 293 cells transfected with vector alone, WT EGFR or ΔECD EGFR for 24 hours. g. Western blot analysis of protein samples for pAkt, Akt, pErk1/2, Erk1/2 and EGFR isolated from PC3 cells treated with EGF +/− AEE788 or EGF +/− C225 for 15 minutes similarly as cells used in c and d. h. A549 cells were treated with AEE788 or C225 for 5 days at the indicated concentrations and measured cell number. i. Colony formation assay on PC3 and A549 cells were treated with increasing concentrations of AEE788 or C225 as indicated in 6-well plate and colony formation was counted when the cells reached 80–90% confluence. Data are means of +/− SD of triplicates ( Suppl Figure 1a and 1b ). Asterisk indicates the statistical significance between treated group and DMSO ( P -value ≤ 0.0001).
Figure Legend Snippet: Constitutive activation of EGFR sustains cell proliferation in the absence of ligands a. Serum starved PC3, DU145, A549 and HT-29 cells were tested for EGFR phosphorylation. b. PC3 and A549 cells grown in the absence of serum/ligands were tested for EGFR dimerization (crosslinked by DMP) and phosphorylation. Serum starved A549 and PC3 were treated with EGF +/− C225 at 2.5 ug/ml c. or AEE788 at 2.5 uM d. for 15 minutes and measured for EGFR phosphorylation, EGFR and Actin using Western blot. e. Schematic diagram of WT and extra cellular domain deleted EGFR (ΔECD-EGFR). f. Western blot analysis of protein samples for pEGFR and EGFR (Flag) isolated from HEK 293 cells transfected with vector alone, WT EGFR or ΔECD EGFR for 24 hours. g. Western blot analysis of protein samples for pAkt, Akt, pErk1/2, Erk1/2 and EGFR isolated from PC3 cells treated with EGF +/− AEE788 or EGF +/− C225 for 15 minutes similarly as cells used in c and d. h. A549 cells were treated with AEE788 or C225 for 5 days at the indicated concentrations and measured cell number. i. Colony formation assay on PC3 and A549 cells were treated with increasing concentrations of AEE788 or C225 as indicated in 6-well plate and colony formation was counted when the cells reached 80–90% confluence. Data are means of +/− SD of triplicates ( Suppl Figure 1a and 1b ). Asterisk indicates the statistical significance between treated group and DMSO ( P -value ≤ 0.0001).

Techniques Used: Activation Assay, Western Blot, Isolation, Transfection, Plasmid Preparation, Colony Assay

32) Product Images from "Targeting the tumor-promoting microenvironment in MET-amplified NSCLC cells with a novel inhibitor of pro-HGF activation"

Article Title: Targeting the tumor-promoting microenvironment in MET-amplified NSCLC cells with a novel inhibitor of pro-HGF activation

Journal: Oncotarget

doi: 10.18632/oncotarget.18260

Inhibition of pro-HGF activation overcomes fibroblast-mediated resistance to MET tyrosine kinase inhibition ( A) EBC-1 cells were treated with JNJ38877605 (25 nM) alone or in the presence of conditioned medium (CM) from WI38 fibroblasts (FIB). CM was also prepared from fibroblasts with silenced HGF (HGF −/− FIB), or from fibroblasts cultured with HGF neutralizing antibody (α-HGF Ab) or SRI31215 (10 μM) as indicated. Cell viability was determined by CellTiter Glo ® 72 h after treatment. (B) H1993 cells were treated with JNJ38877605 (25 nM), fibroblast CM (FIB) and SRI31215 (10 μM) as indicated and cell viability was determined after 72 h. (C) Serum-starved EBC-1 cells were treated with JNJ38877605, recombinant HGF (100 nM), FIB and SRI31215 (10 μM) as indicated for 6 hours. Cell lysates were analyzed by immunoblotting for phospho- and total MET, EGFR, Gab1, AKT and ERK 1/2. *, p
Figure Legend Snippet: Inhibition of pro-HGF activation overcomes fibroblast-mediated resistance to MET tyrosine kinase inhibition ( A) EBC-1 cells were treated with JNJ38877605 (25 nM) alone or in the presence of conditioned medium (CM) from WI38 fibroblasts (FIB). CM was also prepared from fibroblasts with silenced HGF (HGF −/− FIB), or from fibroblasts cultured with HGF neutralizing antibody (α-HGF Ab) or SRI31215 (10 μM) as indicated. Cell viability was determined by CellTiter Glo ® 72 h after treatment. (B) H1993 cells were treated with JNJ38877605 (25 nM), fibroblast CM (FIB) and SRI31215 (10 μM) as indicated and cell viability was determined after 72 h. (C) Serum-starved EBC-1 cells were treated with JNJ38877605, recombinant HGF (100 nM), FIB and SRI31215 (10 μM) as indicated for 6 hours. Cell lysates were analyzed by immunoblotting for phospho- and total MET, EGFR, Gab1, AKT and ERK 1/2. *, p

Techniques Used: Inhibition, Activation Assay, Cell Culture, Recombinant

33) Product Images from "Ion channel TRPV1-dependent activation of PTP1B suppresses EGFR-associated intestinal tumorigenesis"

Article Title: Ion channel TRPV1-dependent activation of PTP1B suppresses EGFR-associated intestinal tumorigenesis

Journal: The Journal of Clinical Investigation

doi: 10.1172/JCI72340

TRPV1 regulates EGFR activity through calpain and PTP1B. ( A ) HCT116 cells were transfected with control or TRPV1 plasmid, then pretreated with DMSO or ALLM (10 μM) for 1 hour, followed by EGF (1 ng/ml) stimulation. Total cell lysates were analyzed by Western blotting. Lanes were run on the same gel. ( B ) HCT116 cells transfected with control or TRPV1 plasmid were pretreated with DMSO, the nonselective PTP inhibitor Na 3 VO 4 (10 μM), or the selective PTP1B inhibitor Compound 3 (10 μM) for 1 hour, followed by EGF stimulation and Western blotting. ( C ) Q-PCR was performed for PTP candidates with RNA isolated from WT colon crypts. ( D ) HCT116 cells were treated with control or PTP1B siRNA and transfected with control or TRPV1 plasmid. Cells were then stimulated with EGF (1 ng/ml) and analyzed by Western blotting. Representative results of 2 independent experiments. ( E ) HCT116 cells were transfected with empty vector, WT PTP1B (PTP435), or PTP1B C-terminal truncation mutants (PTP370 and PTP377). Cells were stimulated with EGF (1 ng/ml) and analyzed by Western blotting. Representative results of 3 independent experiments. ( F ) PTP1B expression in intestinal organoids, shown by confocal fluorescent microscopy. ( G ) Expression of PTP1B in murine small intestinal tissue sections. Immunostaining for PTP1B and detection with DAB (brown) was followed by counterstaining with hematoxylin. ( H ) Q-PCR analysis of villus (V1 and V2) and crypt (C) fractions for Trpv1 and Ptpn1 , showing enrichment in the latter. Values are expressed relative to the V1 fraction. Mean ± SEM ( n = 3 per group). * P
Figure Legend Snippet: TRPV1 regulates EGFR activity through calpain and PTP1B. ( A ) HCT116 cells were transfected with control or TRPV1 plasmid, then pretreated with DMSO or ALLM (10 μM) for 1 hour, followed by EGF (1 ng/ml) stimulation. Total cell lysates were analyzed by Western blotting. Lanes were run on the same gel. ( B ) HCT116 cells transfected with control or TRPV1 plasmid were pretreated with DMSO, the nonselective PTP inhibitor Na 3 VO 4 (10 μM), or the selective PTP1B inhibitor Compound 3 (10 μM) for 1 hour, followed by EGF stimulation and Western blotting. ( C ) Q-PCR was performed for PTP candidates with RNA isolated from WT colon crypts. ( D ) HCT116 cells were treated with control or PTP1B siRNA and transfected with control or TRPV1 plasmid. Cells were then stimulated with EGF (1 ng/ml) and analyzed by Western blotting. Representative results of 2 independent experiments. ( E ) HCT116 cells were transfected with empty vector, WT PTP1B (PTP435), or PTP1B C-terminal truncation mutants (PTP370 and PTP377). Cells were stimulated with EGF (1 ng/ml) and analyzed by Western blotting. Representative results of 3 independent experiments. ( F ) PTP1B expression in intestinal organoids, shown by confocal fluorescent microscopy. ( G ) Expression of PTP1B in murine small intestinal tissue sections. Immunostaining for PTP1B and detection with DAB (brown) was followed by counterstaining with hematoxylin. ( H ) Q-PCR analysis of villus (V1 and V2) and crypt (C) fractions for Trpv1 and Ptpn1 , showing enrichment in the latter. Values are expressed relative to the V1 fraction. Mean ± SEM ( n = 3 per group). * P

Techniques Used: Activity Assay, Transfection, Plasmid Preparation, Western Blot, Polymerase Chain Reaction, Isolation, Expressing, Microscopy, Immunostaining

Proposed model for EGFR-TRPV1 crosstalk in IECs. TRPV1 and PTP1B are part of a homeostatic signaling circuit that restrains EGFR-induced epithelial cell proliferation. EGFR kinase activity mediates proproliferative and thus protumorigenic effects in IECs (dashed red arrows). TRPV1 and PTP1B are predominantly expressed in the crypt compartment that contains TA cells (dark blue cells) with active EGFR signaling. Ligand-induced autophosphorylation of the EGFR results in PLC activation, which cleaves PIP 2 , a tonic inhibitor of TRPV1, into diacylglycerol (DAG) and inositol triphosphate (IP 3 ; green arrows). This results in TRPV1 triggering and Ca 2+ influx, which activates calpain and subsequently PTP1B. PTP1B then dephosphorylates EGFR (blue lines). This coupling between EGFR and TRPV1 exerts negative feedback on growth factor receptor signaling, inhibits crypt progenitor cell (dark blue cells) turnover, and hence reduces the risk of intestinal neoplasia development (red cells). +4 ISC, ISC at +4 position; CBCC, crypt-based columnar stem cell.
Figure Legend Snippet: Proposed model for EGFR-TRPV1 crosstalk in IECs. TRPV1 and PTP1B are part of a homeostatic signaling circuit that restrains EGFR-induced epithelial cell proliferation. EGFR kinase activity mediates proproliferative and thus protumorigenic effects in IECs (dashed red arrows). TRPV1 and PTP1B are predominantly expressed in the crypt compartment that contains TA cells (dark blue cells) with active EGFR signaling. Ligand-induced autophosphorylation of the EGFR results in PLC activation, which cleaves PIP 2 , a tonic inhibitor of TRPV1, into diacylglycerol (DAG) and inositol triphosphate (IP 3 ; green arrows). This results in TRPV1 triggering and Ca 2+ influx, which activates calpain and subsequently PTP1B. PTP1B then dephosphorylates EGFR (blue lines). This coupling between EGFR and TRPV1 exerts negative feedback on growth factor receptor signaling, inhibits crypt progenitor cell (dark blue cells) turnover, and hence reduces the risk of intestinal neoplasia development (red cells). +4 ISC, ISC at +4 position; CBCC, crypt-based columnar stem cell.

Techniques Used: Activity Assay, Planar Chromatography, Activation Assay

TRPV1 signaling inhibits cell proliferation and EGFR activity in vivo. ( A ) Increased IEC proliferation in colons from naive Trpv1 –/– versus WT mice, as shown by Ki67 immunostaining. ( B ) Increased EGFR Y1068 phosphorylation in Trpv1 –/– IECs was reversed by gefitinib treatment. Mice were treated with 2 doses of gefitinib (50 mg/kg, gavage) with a 6-hour interval, followed by IEC harvesting and Western blotting. Shown are representative results from 3 experiments. ( C ) Immunostaining for p-EGFR Y1068 of distal colon tissues. ( D ) Quantification with ImageJ. 3 representative areas were used for analysis, with 3 mice per group. ( E ) Hyperproliferation of Trpv1 –/– IECs was reversed by EGFR inhibition with gefitinib (50 mg/kg) for 5 consecutive days before tissue harvesting. ( F ) Quantification of Ki67 + cells by ImageJ (ImmunoRatio plugin). ( G ) Capsaicin administration suppressed transcription of EGFR target genes in a TRPV1-dependent manner, comparable to gefitinib. Mice received DMSO, capsaicin (3 mg/kg), gefitinib (50 mg/kg), or capsaicin plus gefitinib by gavage, and IECs were prepared 6 hours later. Shown is Q-PCR analysis for c-Fos or Fosl2 , relative to the respective DMSO control. ( H ) Q-PCR analysis for c-Jun . ( I ) Generation of transgenic TRPV1 IEC mice. ( J ) TRPV1 fl/fl and TRPV1 IEC mice were analyzed for p-EGFR Y1068 by Western blotting. IEC-specific TRPV1 overexpression inhibited constitutive EGFR signaling in IECs. ( K ) IEC-specific TRPV1 overexpression suppressed EGF-induced EGFR Y1068 phosphorylation in IECs. Data are mean ± SEM ( D and F ; n = 3 per group) or mean ± SD ( G and H ; n = 2 per group). * P
Figure Legend Snippet: TRPV1 signaling inhibits cell proliferation and EGFR activity in vivo. ( A ) Increased IEC proliferation in colons from naive Trpv1 –/– versus WT mice, as shown by Ki67 immunostaining. ( B ) Increased EGFR Y1068 phosphorylation in Trpv1 –/– IECs was reversed by gefitinib treatment. Mice were treated with 2 doses of gefitinib (50 mg/kg, gavage) with a 6-hour interval, followed by IEC harvesting and Western blotting. Shown are representative results from 3 experiments. ( C ) Immunostaining for p-EGFR Y1068 of distal colon tissues. ( D ) Quantification with ImageJ. 3 representative areas were used for analysis, with 3 mice per group. ( E ) Hyperproliferation of Trpv1 –/– IECs was reversed by EGFR inhibition with gefitinib (50 mg/kg) for 5 consecutive days before tissue harvesting. ( F ) Quantification of Ki67 + cells by ImageJ (ImmunoRatio plugin). ( G ) Capsaicin administration suppressed transcription of EGFR target genes in a TRPV1-dependent manner, comparable to gefitinib. Mice received DMSO, capsaicin (3 mg/kg), gefitinib (50 mg/kg), or capsaicin plus gefitinib by gavage, and IECs were prepared 6 hours later. Shown is Q-PCR analysis for c-Fos or Fosl2 , relative to the respective DMSO control. ( H ) Q-PCR analysis for c-Jun . ( I ) Generation of transgenic TRPV1 IEC mice. ( J ) TRPV1 fl/fl and TRPV1 IEC mice were analyzed for p-EGFR Y1068 by Western blotting. IEC-specific TRPV1 overexpression inhibited constitutive EGFR signaling in IECs. ( K ) IEC-specific TRPV1 overexpression suppressed EGF-induced EGFR Y1068 phosphorylation in IECs. Data are mean ± SEM ( D and F ; n = 3 per group) or mean ± SD ( G and H ; n = 2 per group). * P

Techniques Used: Activity Assay, In Vivo, Mouse Assay, Immunostaining, Western Blot, Inhibition, Polymerase Chain Reaction, Transgenic Assay, Over Expression

34) Product Images from "CHI3L1 promotes proliferation and improves sensitivity to cetuximab in colon cancer cells by down‐regulating p53. CHI3L1 promotes proliferation and improves sensitivity to cetuximab in colon cancer cells by down‐regulating p53"

Article Title: CHI3L1 promotes proliferation and improves sensitivity to cetuximab in colon cancer cells by down‐regulating p53. CHI3L1 promotes proliferation and improves sensitivity to cetuximab in colon cancer cells by down‐regulating p53

Journal: Journal of Clinical Laboratory Analysis

doi: 10.1002/jcla.23026

Mechanism of Chitinase 3‐like protein 1 (CHI3L1) overexpression affecting cell sensitivity to cetuximab. A, The expression of EGFR and p53 in HCT116‐pCDH and HCT116‐CHI3L1 cells after treatment with cetuximab. B, The expression of EGFR and p53 in SW620‐pCDH and SW620‐CHI3L1 cells after treatment with cetuximab. C, The expression level of EGFR and p53 after transfection of p53 overexpression plasmid in HCT116‐CHI3L1 cells. D, p53 overexpression inhibited cell proliferation. E, Overexpression of p53 decreased the sensitivity of HCT116‐pCDH cell to cetuximab. *, P
Figure Legend Snippet: Mechanism of Chitinase 3‐like protein 1 (CHI3L1) overexpression affecting cell sensitivity to cetuximab. A, The expression of EGFR and p53 in HCT116‐pCDH and HCT116‐CHI3L1 cells after treatment with cetuximab. B, The expression of EGFR and p53 in SW620‐pCDH and SW620‐CHI3L1 cells after treatment with cetuximab. C, The expression level of EGFR and p53 after transfection of p53 overexpression plasmid in HCT116‐CHI3L1 cells. D, p53 overexpression inhibited cell proliferation. E, Overexpression of p53 decreased the sensitivity of HCT116‐pCDH cell to cetuximab. *, P

Techniques Used: Over Expression, Expressing, Transfection, Plasmid Preparation

35) Product Images from "Akt1 interacts with epidermal growth factor receptors and Hedgehog signaling to increase stem/transit amplifying cells in the embryonic mouse cortex"

Article Title: Akt1 interacts with epidermal growth factor receptors and Hedgehog signaling to increase stem/transit amplifying cells in the embryonic mouse cortex

Journal: Developmental neurobiology

doi: 10.1002/dneu.20878

Akt signaling is active in EGFR high precursors at the PSB. Cells in sections of E15 mouse cortex were stained with anti-EGFR (A) and anti-phospho-Akt (pAkt) substrate (B) antibodies. Arrows point to cells that are double-labeled for EGFR and pAkt substrate. 58.9+2.1% of the EGFR high cells at the PSB were double-labeled with pAkt-substrate antibody (n=2 brains). Sections from the same brains were double-labeled with pAkt(Ser473) and EGFR antibodies. 10.3±3.2% of the EGFR high cells at the PSB were also pAkt(Ser473) + . Bar = 50 µm.
Figure Legend Snippet: Akt signaling is active in EGFR high precursors at the PSB. Cells in sections of E15 mouse cortex were stained with anti-EGFR (A) and anti-phospho-Akt (pAkt) substrate (B) antibodies. Arrows point to cells that are double-labeled for EGFR and pAkt substrate. 58.9+2.1% of the EGFR high cells at the PSB were double-labeled with pAkt-substrate antibody (n=2 brains). Sections from the same brains were double-labeled with pAkt(Ser473) and EGFR antibodies. 10.3±3.2% of the EGFR high cells at the PSB were also pAkt(Ser473) + . Bar = 50 µm.

Techniques Used: Staining, Labeling

Schematic illustrating possible mechanisms of interaction between Akt, EGF receptor, and Hh signaling. Akt signaling could be important for promoting the self-renewal of stem/TA cells at several steps during embryonic development. For example, early (E13–14) cortical precursor cells that express elevated/activated Akt interpret a low concentration of Shh as though it were higher, leading to up-regulation of EGF receptor expression. At a later stage (E15–16) when the EGFR is expressed at a high level, Akt signaling could act downstream of EGFR activation to promote self-renewal. Precursors from EGFR-null, smoothened-null, and EGFR kinase-inhibited precursors all lack sufficient EGFR activity to allow Akt signaling to promote self-renewal.
Figure Legend Snippet: Schematic illustrating possible mechanisms of interaction between Akt, EGF receptor, and Hh signaling. Akt signaling could be important for promoting the self-renewal of stem/TA cells at several steps during embryonic development. For example, early (E13–14) cortical precursor cells that express elevated/activated Akt interpret a low concentration of Shh as though it were higher, leading to up-regulation of EGF receptor expression. At a later stage (E15–16) when the EGFR is expressed at a high level, Akt signaling could act downstream of EGFR activation to promote self-renewal. Precursors from EGFR-null, smoothened-null, and EGFR kinase-inhibited precursors all lack sufficient EGFR activity to allow Akt signaling to promote self-renewal.

Techniques Used: Concentration Assay, Expressing, Activated Clotting Time Assay, Activation Assay, Activity Assay

Elevated Akt1 does not increase EGFR expression (A) or generation of primary neurospheres (B) by precursors from smoothened mutant cortex. Loss of these responses was observed even in smoothened heterozygous cells. Explants of E12.5–E13.5 smoothened wild type (Emx1 cre/+ ; smo +/+ ), heterozygous (Emx1 cre/+ ; smo flox/+ ), and null (Emx1 cre/+ ; smo flox/− ) cortex were infected with control or Akt1 viruses and assayed 5 days later for expression of EGF receptors and generation of neurospheres in response to EGF (10 ng/ml). N=5 experiments for panel A, N = 5–9 for panel B. * p = 0.02, **.p≤0.0005. (C) Sections of wild-type and smoothened-null E16 cortex were stained with anti-pAkt substrate antibody to assess the status of Akt signaling. No significant difference was observed in smoothened-null cortex with respect to the number of pAkt substrate + precursors (VZ + SVZ) in a 150 µm wide × 100 µm deep region near the PSB at a mid-anterior/posterior level (46.9±1.6 pAkt substrate + cells in wild-type, compared to 35.9±8.9 in smoothened-null cortex, n=2 brains per genotype). LV, lateral ventricle; LGE, lateral ganglionic eminence. Bar = 25 µm.
Figure Legend Snippet: Elevated Akt1 does not increase EGFR expression (A) or generation of primary neurospheres (B) by precursors from smoothened mutant cortex. Loss of these responses was observed even in smoothened heterozygous cells. Explants of E12.5–E13.5 smoothened wild type (Emx1 cre/+ ; smo +/+ ), heterozygous (Emx1 cre/+ ; smo flox/+ ), and null (Emx1 cre/+ ; smo flox/− ) cortex were infected with control or Akt1 viruses and assayed 5 days later for expression of EGF receptors and generation of neurospheres in response to EGF (10 ng/ml). N=5 experiments for panel A, N = 5–9 for panel B. * p = 0.02, **.p≤0.0005. (C) Sections of wild-type and smoothened-null E16 cortex were stained with anti-pAkt substrate antibody to assess the status of Akt signaling. No significant difference was observed in smoothened-null cortex with respect to the number of pAkt substrate + precursors (VZ + SVZ) in a 150 µm wide × 100 µm deep region near the PSB at a mid-anterior/posterior level (46.9±1.6 pAkt substrate + cells in wild-type, compared to 35.9±8.9 in smoothened-null cortex, n=2 brains per genotype). LV, lateral ventricle; LGE, lateral ganglionic eminence. Bar = 25 µm.

Techniques Used: Expressing, Mutagenesis, Infection, Staining

36) Product Images from "Asporin enhances colorectal cancer metastasis through activating the EGFR/Src/cortactin signaling pathway"

Article Title: Asporin enhances colorectal cancer metastasis through activating the EGFR/Src/cortactin signaling pathway

Journal: Oncotarget

doi: 10.18632/oncotarget.12336

Asporin activates cortactin in CRC cells ( A – B ) Western blotting analysis of EGFR, phosphorylated EGFR, Src, phosphorylated Src, cortactin and phosphorylated cortactin in the cytosolic fraction. Serum-starved cells were stimulated with EGF (10 ng/ml) in the presence of PP2 (10 μM). GAPDH was used as a loading control.
Figure Legend Snippet: Asporin activates cortactin in CRC cells ( A – B ) Western blotting analysis of EGFR, phosphorylated EGFR, Src, phosphorylated Src, cortactin and phosphorylated cortactin in the cytosolic fraction. Serum-starved cells were stimulated with EGF (10 ng/ml) in the presence of PP2 (10 μM). GAPDH was used as a loading control.

Techniques Used: Western Blot

37) Product Images from "Glycerol kinase 5 confers gefitinib resistance through SREBP1/SCD1 signaling pathway"

Article Title: Glycerol kinase 5 confers gefitinib resistance through SREBP1/SCD1 signaling pathway

Journal: Journal of Experimental & Clinical Cancer Research : CR

doi: 10.1186/s13046-019-1057-7

GK5 knockdown suppresses PC9R cell proliferation by inhibiting EGFR/AKT/SREBP1/SCD1 signaling molecules. a Whole human cDNA array analysis. b Western blotting on protein levels of EGFR, p-AKT, AKT, survivin, PLK1, SREBP1, SCD1 and Bcl-2 in PC9R cells c CCK8 assay on cell proliferation of PC9R cells overexpressing EGFR and infected with viral particles expressing shGK5–1 and shNEG. d CCK8 assay on cell proliferation of PC9R cells transfected with shGK5–1, − 2 or shNEG and treated with YM155. * p
Figure Legend Snippet: GK5 knockdown suppresses PC9R cell proliferation by inhibiting EGFR/AKT/SREBP1/SCD1 signaling molecules. a Whole human cDNA array analysis. b Western blotting on protein levels of EGFR, p-AKT, AKT, survivin, PLK1, SREBP1, SCD1 and Bcl-2 in PC9R cells c CCK8 assay on cell proliferation of PC9R cells overexpressing EGFR and infected with viral particles expressing shGK5–1 and shNEG. d CCK8 assay on cell proliferation of PC9R cells transfected with shGK5–1, − 2 or shNEG and treated with YM155. * p

Techniques Used: Western Blot, CCK-8 Assay, Infection, Expressing, Transfection

GK5 mediates gefitinib resistance. a Total Internal Reflection Fluorescence (TIRF) Microscopy images of exosomes isolated from plasma of EGFR TKI-sensitive and -resistant patients. b Exosomal GK5 mRNA in the plasma of gefitinib-sensitive and -resistant lung adenocarcinoma patients measured using TCLN biochips. c , d The RT-PCR and Western blotting on GK5 expression in gefitinib-sensitive PC9 and gefitinib-resistant PC9R cells. * p
Figure Legend Snippet: GK5 mediates gefitinib resistance. a Total Internal Reflection Fluorescence (TIRF) Microscopy images of exosomes isolated from plasma of EGFR TKI-sensitive and -resistant patients. b Exosomal GK5 mRNA in the plasma of gefitinib-sensitive and -resistant lung adenocarcinoma patients measured using TCLN biochips. c , d The RT-PCR and Western blotting on GK5 expression in gefitinib-sensitive PC9 and gefitinib-resistant PC9R cells. * p

Techniques Used: Fluorescence, Microscopy, Isolation, Reverse Transcription Polymerase Chain Reaction, Western Blot, Expressing

38) Product Images from "FOXA1, GATA3 and PPARɣ Cooperate to Drive Luminal Subtype in Bladder Cancer: A Molecular Analysis of Established Human Cell Lines"

Article Title: FOXA1, GATA3 and PPARɣ Cooperate to Drive Luminal Subtype in Bladder Cancer: A Molecular Analysis of Established Human Cell Lines

Journal: Scientific Reports

doi: 10.1038/srep38531

In vivo experiments show a subset of human cancer cell lines exhibits histomorphology and marker expression consistent with molecular subtype assignment. Hematoxylin and eosin stain (top panels) and immunohistochemistry for the luminal markers FOXA1, GATA3, and UPK, as well as the basal markers KRT5/6, KRT14, and EGFR following tissue recombination of ( A ) RT4, ( B ) UMUC1, and ( C ) SCaBER human bladder cancer cell lines.
Figure Legend Snippet: In vivo experiments show a subset of human cancer cell lines exhibits histomorphology and marker expression consistent with molecular subtype assignment. Hematoxylin and eosin stain (top panels) and immunohistochemistry for the luminal markers FOXA1, GATA3, and UPK, as well as the basal markers KRT5/6, KRT14, and EGFR following tissue recombination of ( A ) RT4, ( B ) UMUC1, and ( C ) SCaBER human bladder cancer cell lines.

Techniques Used: In Vivo, Marker, Expressing, H&E Stain, Immunohistochemistry

Candidate gene analysis suggests GATA3 and FOXA1 cooperate with PPARɣ activation to regulate molecular subtype-specific gene expression in bladder cancer cells. ( A ) Western blotting analysis of a panel of human bladder cancer cell lines for expression of FOXA1 and PPARɣ. Cells are arranged by molecular subtype based on analysis presented in Fig. 1 . ( B ) Q-RT-PCR analysis of human bladder cancer cell lines for GATA3. ( C–F ) Q-RT-PCR analysis for the luminal bladder cancer markers ( C ) FGFR3 and ( D ) KRT20, as well as the basal bladder cancer markers ( E ) EGFR and ( F ) KRT6 following individual overexpression of empty vector (CMV), FOXA1, GATA3, or FOXA1 and GATA3 in the presence of vehicle control (DMSO) or 1 micromolar rosiglitazone (TZD). See materials and methods for detailed description of experimental approach.
Figure Legend Snippet: Candidate gene analysis suggests GATA3 and FOXA1 cooperate with PPARɣ activation to regulate molecular subtype-specific gene expression in bladder cancer cells. ( A ) Western blotting analysis of a panel of human bladder cancer cell lines for expression of FOXA1 and PPARɣ. Cells are arranged by molecular subtype based on analysis presented in Fig. 1 . ( B ) Q-RT-PCR analysis of human bladder cancer cell lines for GATA3. ( C–F ) Q-RT-PCR analysis for the luminal bladder cancer markers ( C ) FGFR3 and ( D ) KRT20, as well as the basal bladder cancer markers ( E ) EGFR and ( F ) KRT6 following individual overexpression of empty vector (CMV), FOXA1, GATA3, or FOXA1 and GATA3 in the presence of vehicle control (DMSO) or 1 micromolar rosiglitazone (TZD). See materials and methods for detailed description of experimental approach.

Techniques Used: Activation Assay, Expressing, Western Blot, Reverse Transcription Polymerase Chain Reaction, Over Expression, Plasmid Preparation

39) Product Images from "CircHIPK3 promotes colorectal cancer growth and metastasis by sponging miR-7"

Article Title: CircHIPK3 promotes colorectal cancer growth and metastasis by sponging miR-7

Journal: Cell Death & Disease

doi: 10.1038/s41419-018-0454-8

Overexpression of circHIPK3 effectively reverses miR-7-induced inhibition of CRC cells progression. HCT116 and HT29 cells transfected with miR-control, miR-7, circHIPK3, or miR-7 + circHIPK3. Then the ability of cell cloning, proliferation, migration and invasion was, respectively, assessed by colony formation assay ( a ), EdU assay ( b ), transwell migration ( d ), and matrigel invasion ( e ) assay. And cell apoptosis analysis was tested by flow cytometry with Annexin V-FITC/PI double staining ( c ). * vs control group, # vs mir-7 group. f qRT-PCR analysis of the expression of growth and metastasis-related miR-7 targets in HCT116 and HT29 cells with circHIPK3 knockdown or overexpression. * vs si-NC group, # vs vector group. g Western blot analysis of the protein expression of FAK, IGF1R, EGFR, and YY1 in fore-mentioned four groups in HCT116 and HT29 cells. Data were represented as means ± S.D. of at least three independent experiments. * p
Figure Legend Snippet: Overexpression of circHIPK3 effectively reverses miR-7-induced inhibition of CRC cells progression. HCT116 and HT29 cells transfected with miR-control, miR-7, circHIPK3, or miR-7 + circHIPK3. Then the ability of cell cloning, proliferation, migration and invasion was, respectively, assessed by colony formation assay ( a ), EdU assay ( b ), transwell migration ( d ), and matrigel invasion ( e ) assay. And cell apoptosis analysis was tested by flow cytometry with Annexin V-FITC/PI double staining ( c ). * vs control group, # vs mir-7 group. f qRT-PCR analysis of the expression of growth and metastasis-related miR-7 targets in HCT116 and HT29 cells with circHIPK3 knockdown or overexpression. * vs si-NC group, # vs vector group. g Western blot analysis of the protein expression of FAK, IGF1R, EGFR, and YY1 in fore-mentioned four groups in HCT116 and HT29 cells. Data were represented as means ± S.D. of at least three independent experiments. * p

Techniques Used: Over Expression, Inhibition, Transfection, Clone Assay, Migration, Colony Assay, EdU Assay, Flow Cytometry, Cytometry, Double Staining, Quantitative RT-PCR, Expressing, Plasmid Preparation, Western Blot

The schematic cartoon of the c-Myb/circHIPK3/miR-7 axis in CRC. In CRC, circHIPK3 is frequently upregulated partly caused by the transcription factor c-Myb overexpression, then circHIPK3 can sponge more endogenous miR-7 to sequester and inhibit miR-7 activity, thereby leading to increased proto-oncogenes (FAK, IGF1R, EGFR, and YY1) expression, which promoting CRC development and progression
Figure Legend Snippet: The schematic cartoon of the c-Myb/circHIPK3/miR-7 axis in CRC. In CRC, circHIPK3 is frequently upregulated partly caused by the transcription factor c-Myb overexpression, then circHIPK3 can sponge more endogenous miR-7 to sequester and inhibit miR-7 activity, thereby leading to increased proto-oncogenes (FAK, IGF1R, EGFR, and YY1) expression, which promoting CRC development and progression

Techniques Used: Over Expression, Activity Assay, Expressing

40) Product Images from "ANO1/TMEM16A interacts with EGFR and correlates with sensitivity to EGFR-targeting therapy in head and neck cancer"

Article Title: ANO1/TMEM16A interacts with EGFR and correlates with sensitivity to EGFR-targeting therapy in head and neck cancer

Journal: Oncotarget

doi:

Interaction between ANO1 and EGFR involves the trans/juxtamembrane domain of EGFR (A) Schematic of the EGFR-constructs tested for interaction with ANO1. (B) Immunoprecipitation of ANO1 and FLAG-tagged truncation variants of lz-EGFR in HEK293T cell lysates. HEK293T cells were transfected with equal amounts of plasmids encoding ANO1 and lz-EGFR-variants. EGFR/ANO1 complexes were analyzed by immunoprecipitation using an anti-ANO1 or anti-FLAG antibody coupled to magnetic beads and immunoblotting of the eluted proteins. Representative immunoblots are shown. (C) Immunoprecipitation of lz-EGFR and ANO1 truncation variants in HEK293T cell lysates. HEK293T cells were transfected with equal amounts of plasmids encoding lz-EGFR and ANO1-variants and ANO1/lz-EGFR complexes were analyzed as in Figure 2B . The multiple bands for ANO1 represent different glycosylation variants of ANO1 [ 39 ].
Figure Legend Snippet: Interaction between ANO1 and EGFR involves the trans/juxtamembrane domain of EGFR (A) Schematic of the EGFR-constructs tested for interaction with ANO1. (B) Immunoprecipitation of ANO1 and FLAG-tagged truncation variants of lz-EGFR in HEK293T cell lysates. HEK293T cells were transfected with equal amounts of plasmids encoding ANO1 and lz-EGFR-variants. EGFR/ANO1 complexes were analyzed by immunoprecipitation using an anti-ANO1 or anti-FLAG antibody coupled to magnetic beads and immunoblotting of the eluted proteins. Representative immunoblots are shown. (C) Immunoprecipitation of lz-EGFR and ANO1 truncation variants in HEK293T cell lysates. HEK293T cells were transfected with equal amounts of plasmids encoding lz-EGFR and ANO1-variants and ANO1/lz-EGFR complexes were analyzed as in Figure 2B . The multiple bands for ANO1 represent different glycosylation variants of ANO1 [ 39 ].

Techniques Used: Construct, Immunoprecipitation, Transfection, Magnetic Beads, Western Blot

Expression of ANO1 predicts susceptibility to Gefitinib in HNSCC cell lines (A) Relative mRNA-levels of ANO1 and EGFR (bars, left y-axis) and sensitivity to Gefitinib (IC50, circles, right y-axis) of HNSCC cell lines, determined by quantitative PCR and Cell Titer Glo, respectively. A Pearson-correlation test was used to test for correlation between ANO1/EGFR expression and sensitivity to Gefitinib. (B) ANO1 protein levels in the HNSCC cell lines shown in Figure 5A .
Figure Legend Snippet: Expression of ANO1 predicts susceptibility to Gefitinib in HNSCC cell lines (A) Relative mRNA-levels of ANO1 and EGFR (bars, left y-axis) and sensitivity to Gefitinib (IC50, circles, right y-axis) of HNSCC cell lines, determined by quantitative PCR and Cell Titer Glo, respectively. A Pearson-correlation test was used to test for correlation between ANO1/EGFR expression and sensitivity to Gefitinib. (B) ANO1 protein levels in the HNSCC cell lines shown in Figure 5A .

Techniques Used: Expressing, Real-time Polymerase Chain Reaction

ANO1 and EGFR form a complex in HNSCC cells (A) Summary of discovery proteomics experiments after ANO1-pulldown in Te11 cells. ANO1 was immunoprecipitated from Te11 cell lysates and proteins co-purified with ANO1 were analyzed by LC-MS. 40 proteins were identified to interact with ANO1 in all three experiments with a C-score
Figure Legend Snippet: ANO1 and EGFR form a complex in HNSCC cells (A) Summary of discovery proteomics experiments after ANO1-pulldown in Te11 cells. ANO1 was immunoprecipitated from Te11 cell lysates and proteins co-purified with ANO1 were analyzed by LC-MS. 40 proteins were identified to interact with ANO1 in all three experiments with a C-score

Techniques Used: Immunoprecipitation, Purification, Liquid Chromatography with Mass Spectroscopy

EGFR and ANO1 regulate each other's protein levels (A) Immunoblot of EGFR, phospho-EGFR and ANO1 protein levels in Te11 cells stably expressing dox-inducible expression constructs for EGFR-wt, -D837A, lz-EGFR or lz-EGFR-D837A or an empty vector control, in the presence or absence of dox (48 h) and Gefitinib (1 μM, 24 h). Tubulin served as a loading control. Representative immunoblots are shown. (B) Relative mRNA levels of EGFR and ANO1 in the same samples as used in A. mRNA-levels in dox-treated samples were normalized to the respective non-dox treated sample and are presented as the mean ± SEM of three independent experiments. (C) Relative cell proliferation of Te11 cells stably expressing the indicated dox-inducible constructs analyzed by Cell Titer Glo. Signals were normalized to the respective non-dox treated sample and are presented as the mean ± SEM of four independent experiments, p
Figure Legend Snippet: EGFR and ANO1 regulate each other's protein levels (A) Immunoblot of EGFR, phospho-EGFR and ANO1 protein levels in Te11 cells stably expressing dox-inducible expression constructs for EGFR-wt, -D837A, lz-EGFR or lz-EGFR-D837A or an empty vector control, in the presence or absence of dox (48 h) and Gefitinib (1 μM, 24 h). Tubulin served as a loading control. Representative immunoblots are shown. (B) Relative mRNA levels of EGFR and ANO1 in the same samples as used in A. mRNA-levels in dox-treated samples were normalized to the respective non-dox treated sample and are presented as the mean ± SEM of three independent experiments. (C) Relative cell proliferation of Te11 cells stably expressing the indicated dox-inducible constructs analyzed by Cell Titer Glo. Signals were normalized to the respective non-dox treated sample and are presented as the mean ± SEM of four independent experiments, p

Techniques Used: Stable Transfection, Expressing, Construct, Plasmid Preparation, Western Blot

EGFR and ANO1 form a functional complex which regulates cancer cell proliferation (A) Immunoblots of EGFR, phospho-EGFR (Y1068) and ANO1 protein levels in Te11 cells stably co-expressing dox-inducible shRNAs against ANO1 and dox-inducible expression constructs for EGFR-wt, lz-EGFR or an empty vector control after treatment with dox for 72 h. Representative immunoblots are shown. (B) Relative mRNA-levels of ANO1 and EGFR in Te11 cells treated as in A. mRNA-levels in dox-treated samples were normalized to the respective non-dox treated sample and are presented as the mean ± SEM of three independent experiments. (C) Colony formation assay of Te11 cells stably co-expressing dox-inducible shRNAs against ANO1 and dox-inducible expression constructs for EGFR-wt, lz-EGFR or an empty vector control. Representative images are shown. (D) Quantification of the relative colony area of Te11 cells treated as in C. Values were normalized to the respective non-dox treated sample and are presented as the mean ± SEM of three independent experiments. ( p
Figure Legend Snippet: EGFR and ANO1 form a functional complex which regulates cancer cell proliferation (A) Immunoblots of EGFR, phospho-EGFR (Y1068) and ANO1 protein levels in Te11 cells stably co-expressing dox-inducible shRNAs against ANO1 and dox-inducible expression constructs for EGFR-wt, lz-EGFR or an empty vector control after treatment with dox for 72 h. Representative immunoblots are shown. (B) Relative mRNA-levels of ANO1 and EGFR in Te11 cells treated as in A. mRNA-levels in dox-treated samples were normalized to the respective non-dox treated sample and are presented as the mean ± SEM of three independent experiments. (C) Colony formation assay of Te11 cells stably co-expressing dox-inducible shRNAs against ANO1 and dox-inducible expression constructs for EGFR-wt, lz-EGFR or an empty vector control. Representative images are shown. (D) Quantification of the relative colony area of Te11 cells treated as in C. Values were normalized to the respective non-dox treated sample and are presented as the mean ± SEM of three independent experiments. ( p

Techniques Used: Functional Assay, Western Blot, Stable Transfection, Expressing, Construct, Plasmid Preparation, Colony Assay

Related Articles

Mutagenesis:

Article Title: A Comparative Study of Cell Block versus Biopsy for Detection of Epidermal Growth Factor Receptor Mutations and Anaplastic Lymphoma Kinase Rearrangement in Adenocarcinoma Lung
Article Snippet: .. EGFR IHC was performed using EGFR receptor (E746-A750 del specific) (6B6) XP™ rabbit monoclonal antibody (cell signalling, Danvers, MA) and EGFR receptor (L858R Mutant Specific) (43B2) rabbit monoclonal antibody (cell signalling, Danvers, MA). .. The positive controls used were cases diagnosed to have the E746A750 or L858R mutations by real-time PCR.

Western Blot:

Article Title: Specific Inhibition of Tumor Cells by Oncogenic EGFR Specific Silencing by RNA interference
Article Snippet: .. The primary antibodies used in Western blotting and their dilution ratios in parentheses were as follows: anti-epidermal growth factor receptor (EGFR) (1/1000), anti-phospho-EGFR (1/1000), anti-EGFR (E746-A750del Specific) (6B6) (1/1000), anti-Akt (1/1000), anti-phospho-Akt (1/1000), anti-Erk1/2 (1/2000), and anti-phospho-Erk1/2 (1/2000) antibodies; all of these antibodies were purchased from Cell Signaling Technology. .. The anti-Tubulin antibody (1/10000) was purchased from Sigma Aldrich.

Immunohistochemistry:

Article Title: A Comparative Study of Cell Block versus Biopsy for Detection of Epidermal Growth Factor Receptor Mutations and Anaplastic Lymphoma Kinase Rearrangement in Adenocarcinoma Lung
Article Snippet: .. EGFR IHC was performed using EGFR receptor (E746-A750 del specific) (6B6) XP™ rabbit monoclonal antibody (cell signalling, Danvers, MA) and EGFR receptor (L858R Mutant Specific) (43B2) rabbit monoclonal antibody (cell signalling, Danvers, MA). .. The positive controls used were cases diagnosed to have the E746A750 or L858R mutations by real-time PCR.

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    Cell Signaling Technology Inc anti egfr antibody
    Validation of specific peptide binding to <t>EGFR.</t> On confocal microscopy, we found strong binding of ( a ) <t>QRH*-Cy5.5</t> peptide (red) and ( b ) AF488-labeled anti-EGFR (green) to the surface (arrow) of control HT29 cells (siCL). ( c ) PEH*-Cy5.5 (red) binding is minimal. ( d – f ) The fluorescence intensities are significantly reduced in knockdown of HT29 cells (siEGFR). ( g ) Quantified results for QRH*-Cy5.5 and anti-EGFR show significantly higher intensities for siCL- vs. siEGFR-transfected cells (3.2- and 3.4-fold change, P =0.0021 and 0.0017, respectively), whereas PEH*-Cy5.5 showed a nonsignificant decrease (0.87 fold-change, P =0.57). Differences for siCL vs. siEGFR for QRH*-Cy5.5 and anti-EGFR were significantly greater than those for PEH*-Cy5.5 ( P =0.007 and 0.006, respectively). We fit two-way ANOVA models with the terms for six conditions and two replicate slides on log-transformed data. Measurements were on an average of five randomly chosen cells on two slides for each condition. ( h ) Western blot shows EGFR expression levels. ( i ) On competition, we found a significant difference in binding of QRH*-Cy5.5 to HT29 cells with the addition of unlabeled QRH* and PEH* at the concentrations of 50 μ m and higher. Nonsignficant difference was found at 0 μ m . We fit two-way ANOVA models with the terms for the labeled peptide, concentrations of the unlabeled peptides, and their interactions on log-transformed data. P -values shown here compare the difference in the intensity between unlabeled QRH* and PEH* at each dose with that at 0 μ m . Measurements are on an average of five randomly chosen cells on two slides for each condition. ANOVA, analysis of variance; EGFR, epidermal growth factor receptor.
    Anti Egfr Antibody, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 93/100, based on 26 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Validation of specific peptide binding to EGFR. On confocal microscopy, we found strong binding of ( a ) QRH*-Cy5.5 peptide (red) and ( b ) AF488-labeled anti-EGFR (green) to the surface (arrow) of control HT29 cells (siCL). ( c ) PEH*-Cy5.5 (red) binding is minimal. ( d – f ) The fluorescence intensities are significantly reduced in knockdown of HT29 cells (siEGFR). ( g ) Quantified results for QRH*-Cy5.5 and anti-EGFR show significantly higher intensities for siCL- vs. siEGFR-transfected cells (3.2- and 3.4-fold change, P =0.0021 and 0.0017, respectively), whereas PEH*-Cy5.5 showed a nonsignificant decrease (0.87 fold-change, P =0.57). Differences for siCL vs. siEGFR for QRH*-Cy5.5 and anti-EGFR were significantly greater than those for PEH*-Cy5.5 ( P =0.007 and 0.006, respectively). We fit two-way ANOVA models with the terms for six conditions and two replicate slides on log-transformed data. Measurements were on an average of five randomly chosen cells on two slides for each condition. ( h ) Western blot shows EGFR expression levels. ( i ) On competition, we found a significant difference in binding of QRH*-Cy5.5 to HT29 cells with the addition of unlabeled QRH* and PEH* at the concentrations of 50 μ m and higher. Nonsignficant difference was found at 0 μ m . We fit two-way ANOVA models with the terms for the labeled peptide, concentrations of the unlabeled peptides, and their interactions on log-transformed data. P -values shown here compare the difference in the intensity between unlabeled QRH* and PEH* at each dose with that at 0 μ m . Measurements are on an average of five randomly chosen cells on two slides for each condition. ANOVA, analysis of variance; EGFR, epidermal growth factor receptor.

    Journal: Clinical and Translational Gastroenterology

    Article Title: EGFR Overexpressed in Colonic Neoplasia Can be Detected on Wide-Field Endoscopic Imaging

    doi: 10.1038/ctg.2015.28

    Figure Lengend Snippet: Validation of specific peptide binding to EGFR. On confocal microscopy, we found strong binding of ( a ) QRH*-Cy5.5 peptide (red) and ( b ) AF488-labeled anti-EGFR (green) to the surface (arrow) of control HT29 cells (siCL). ( c ) PEH*-Cy5.5 (red) binding is minimal. ( d – f ) The fluorescence intensities are significantly reduced in knockdown of HT29 cells (siEGFR). ( g ) Quantified results for QRH*-Cy5.5 and anti-EGFR show significantly higher intensities for siCL- vs. siEGFR-transfected cells (3.2- and 3.4-fold change, P =0.0021 and 0.0017, respectively), whereas PEH*-Cy5.5 showed a nonsignificant decrease (0.87 fold-change, P =0.57). Differences for siCL vs. siEGFR for QRH*-Cy5.5 and anti-EGFR were significantly greater than those for PEH*-Cy5.5 ( P =0.007 and 0.006, respectively). We fit two-way ANOVA models with the terms for six conditions and two replicate slides on log-transformed data. Measurements were on an average of five randomly chosen cells on two slides for each condition. ( h ) Western blot shows EGFR expression levels. ( i ) On competition, we found a significant difference in binding of QRH*-Cy5.5 to HT29 cells with the addition of unlabeled QRH* and PEH* at the concentrations of 50 μ m and higher. Nonsignficant difference was found at 0 μ m . We fit two-way ANOVA models with the terms for the labeled peptide, concentrations of the unlabeled peptides, and their interactions on log-transformed data. P -values shown here compare the difference in the intensity between unlabeled QRH* and PEH* at each dose with that at 0 μ m . Measurements are on an average of five randomly chosen cells on two slides for each condition. ANOVA, analysis of variance; EGFR, epidermal growth factor receptor.

    Article Snippet: QRH*-Cy5.5 and anti-EGFR antibody showed significantly higher intensities for siCL-transfected HT29 cells than for those treated with siEGFR, whereas PEH*-Cy5.5 showed a small nonsignificant increase, .

    Techniques: Binding Assay, Confocal Microscopy, Labeling, Fluorescence, Transfection, Transformation Assay, Western Blot, Expressing

    Characterization of EGFR peptide-binding parameters. ( a ) Apparent dissociation constant k d =50 n m , R 2 =0.95 was measured for binding of QRH*-Cy5.5 to HT29 cells. ( b ) Apparent association time constant k =0.406/min (2.46 min) was measured for binding of QRH*-Cy5.5 to HT29 cells. Both results are representative of six independent experiments.

    Journal: Clinical and Translational Gastroenterology

    Article Title: EGFR Overexpressed in Colonic Neoplasia Can be Detected on Wide-Field Endoscopic Imaging

    doi: 10.1038/ctg.2015.28

    Figure Lengend Snippet: Characterization of EGFR peptide-binding parameters. ( a ) Apparent dissociation constant k d =50 n m , R 2 =0.95 was measured for binding of QRH*-Cy5.5 to HT29 cells. ( b ) Apparent association time constant k =0.406/min (2.46 min) was measured for binding of QRH*-Cy5.5 to HT29 cells. Both results are representative of six independent experiments.

    Article Snippet: QRH*-Cy5.5 and anti-EGFR antibody showed significantly higher intensities for siCL-transfected HT29 cells than for those treated with siEGFR, whereas PEH*-Cy5.5 showed a small nonsignificant increase, .

    Techniques: Binding Assay

    Peptide specific for EGFR. ( a ) Chemical structure of QRHKPRE peptide (black) with GGGSK linker (blue) and Cy5.5 fluorophore (red). ( b ) Scrambled peptide PEHKRRQ (control). ( c ) QRH*-Cy5.5 was found on the structural model to bind domain 2 of EGFR (1IVO). ( d ) Fluorescence spectra of Cy5.5-labeled peptides with λ ex =671 nm shows peak emission near 710 nm. AU, arbitrary unit; EGFR, epidermal growth factor receptor.

    Journal: Clinical and Translational Gastroenterology

    Article Title: EGFR Overexpressed in Colonic Neoplasia Can be Detected on Wide-Field Endoscopic Imaging

    doi: 10.1038/ctg.2015.28

    Figure Lengend Snippet: Peptide specific for EGFR. ( a ) Chemical structure of QRHKPRE peptide (black) with GGGSK linker (blue) and Cy5.5 fluorophore (red). ( b ) Scrambled peptide PEHKRRQ (control). ( c ) QRH*-Cy5.5 was found on the structural model to bind domain 2 of EGFR (1IVO). ( d ) Fluorescence spectra of Cy5.5-labeled peptides with λ ex =671 nm shows peak emission near 710 nm. AU, arbitrary unit; EGFR, epidermal growth factor receptor.

    Article Snippet: QRH*-Cy5.5 and anti-EGFR antibody showed significantly higher intensities for siCL-transfected HT29 cells than for those treated with siEGFR, whereas PEH*-Cy5.5 showed a small nonsignificant increase, .

    Techniques: Fluorescence, Labeling

    Binding of EGFR peptide and antibody to human colonic neoplasia. On confocal microscopy, binding of ( a ) QRH*-Cy5.5 peptide (red) co-localizes with that of ( b ) AF488-labeled anti-EGFR antibody (green) on surface of dysplastic colonocytes (arrow), shown in ( c ) merged image, P= 0.71. ( d ) Image contrast can be appreciated at lesion border. Magnified view of boxes in d is shown for ( e ) dysplasia and ( f ) normal. ( g ) Corresponding immunohistochemistry from a shows increased reactivity for EGFR in dysplasia. ( h ) Dysplasia ( n =29) showed significantly higher fluorescence intensities than normal ( n =15) by an average of 19.4-fold, P =1.7 × 10 −9 by two-sample t -test on log-transformed data. ( i ) Receiver operating characteristic curve shows 90% sensitivity and 93% specificity with area under curve (AUC) of 0.94 for distinguishing dysplasia from normal using peptide.

    Journal: Clinical and Translational Gastroenterology

    Article Title: EGFR Overexpressed in Colonic Neoplasia Can be Detected on Wide-Field Endoscopic Imaging

    doi: 10.1038/ctg.2015.28

    Figure Lengend Snippet: Binding of EGFR peptide and antibody to human colonic neoplasia. On confocal microscopy, binding of ( a ) QRH*-Cy5.5 peptide (red) co-localizes with that of ( b ) AF488-labeled anti-EGFR antibody (green) on surface of dysplastic colonocytes (arrow), shown in ( c ) merged image, P= 0.71. ( d ) Image contrast can be appreciated at lesion border. Magnified view of boxes in d is shown for ( e ) dysplasia and ( f ) normal. ( g ) Corresponding immunohistochemistry from a shows increased reactivity for EGFR in dysplasia. ( h ) Dysplasia ( n =29) showed significantly higher fluorescence intensities than normal ( n =15) by an average of 19.4-fold, P =1.7 × 10 −9 by two-sample t -test on log-transformed data. ( i ) Receiver operating characteristic curve shows 90% sensitivity and 93% specificity with area under curve (AUC) of 0.94 for distinguishing dysplasia from normal using peptide.

    Article Snippet: QRH*-Cy5.5 and anti-EGFR antibody showed significantly higher intensities for siCL-transfected HT29 cells than for those treated with siEGFR, whereas PEH*-Cy5.5 showed a small nonsignificant increase, .

    Techniques: Binding Assay, Confocal Microscopy, Labeling, Immunohistochemistry, Fluorescence, Transformation Assay

    Chromatin immunoprecipitation assay and Western blotting analysis of c-Myc, iNOS, Cyclin D1, and VEGF expression in Panc-1 and Colo-357 cells. (A), Agarose gel electrophoresis of the Polymerase Chain Reaction (PCR)-amplified c-Myc gene fragment from the chromatin DNA precipitated with antibody against EGFR, Src, or Stat3, or with the non-specific IgG; and (B and C), Immunoblotting analysis of whole-cell lysates probing for EGFR or Src (B(i) and C(i)) or c-Myc, iNOS, Cyclin D1 or VEGF (B(ii) and C(ii)), and the effects of siRNA knockdown of EGFR (EGFR siRNA), Src (Src siRNA) or control (con) siRNA, or S3I-201 or Das). Bands corresponding to proteins or c-Myc gene in gel are shown; M, molecular weight marker, EGFR/Src, sequential immunoprecipitation with anti-EGFR and then anti-Src antibody. Data are representative of 3 independent studies, and values are mean and s.d of 3 independent studies; * p -

    Journal: PLoS ONE

    Article Title: A Functional Nuclear Epidermal Growth Factor Receptor, Src and Stat3 Heteromeric Complex in Pancreatic Cancer Cells

    doi: 10.1371/journal.pone.0019605

    Figure Lengend Snippet: Chromatin immunoprecipitation assay and Western blotting analysis of c-Myc, iNOS, Cyclin D1, and VEGF expression in Panc-1 and Colo-357 cells. (A), Agarose gel electrophoresis of the Polymerase Chain Reaction (PCR)-amplified c-Myc gene fragment from the chromatin DNA precipitated with antibody against EGFR, Src, or Stat3, or with the non-specific IgG; and (B and C), Immunoblotting analysis of whole-cell lysates probing for EGFR or Src (B(i) and C(i)) or c-Myc, iNOS, Cyclin D1 or VEGF (B(ii) and C(ii)), and the effects of siRNA knockdown of EGFR (EGFR siRNA), Src (Src siRNA) or control (con) siRNA, or S3I-201 or Das). Bands corresponding to proteins or c-Myc gene in gel are shown; M, molecular weight marker, EGFR/Src, sequential immunoprecipitation with anti-EGFR and then anti-Src antibody. Data are representative of 3 independent studies, and values are mean and s.d of 3 independent studies; * p -

    Article Snippet: Compared to anti-Stat3 or anti-Src antibody, the particle size increase upon addition of anti-EGFR antibody to the assay solution is much smaller, only slightly higher than the non-specific rabbit IgG.

    Techniques: Chromatin Immunoprecipitation, Western Blot, Expressing, Agarose Gel Electrophoresis, Polymerase Chain Reaction, Amplification, Molecular Weight, Marker, Immunoprecipitation

    Co-immunoprecipitation with immunoblotting analysis of the effects of modulation of EGFR, Src and Stat3 on the nuclear EGFR, Src and Stat3 complex. (A, B, and C) Immunoblotting analyses of immunecomplexes of Stat3 (IP:Stat3), EGFR (IP:EGFR), or Src (IP:Src) prepared from nuclear extracts of Panc-1 cells untransfected or transfected with Src siRNA, EGFR siRNA, or control (con) siRNA (A), or treated with or without the EGFR inhibitor (ZD1839, ZD), Src inhibitor (Dasatinib, Das), or the Stat3 inhibitor (S3I-201) for 1 or 24 h (B), or from nuclear extracts pre-incubated for 2 h with or without 100 µM pY1068, pY1086, or SPI peptide (C) and probing for EGFR, Src, Stat3; or (D) immunoblotting analysis of nuclear extracts prepared from Panc-1 cells treated or untreated with phenylarsine oxide (PAO) and probing for Src, Stat3, EGFR. Bands corresponding to proteins in gel are shown; input: except where indicated, represents the immunoblotting for the respective immunoprecipitated protein in the same amount of lysate or nuclear extract used in the assay; Data are representative of 3 independent studies.

    Journal: PLoS ONE

    Article Title: A Functional Nuclear Epidermal Growth Factor Receptor, Src and Stat3 Heteromeric Complex in Pancreatic Cancer Cells

    doi: 10.1371/journal.pone.0019605

    Figure Lengend Snippet: Co-immunoprecipitation with immunoblotting analysis of the effects of modulation of EGFR, Src and Stat3 on the nuclear EGFR, Src and Stat3 complex. (A, B, and C) Immunoblotting analyses of immunecomplexes of Stat3 (IP:Stat3), EGFR (IP:EGFR), or Src (IP:Src) prepared from nuclear extracts of Panc-1 cells untransfected or transfected with Src siRNA, EGFR siRNA, or control (con) siRNA (A), or treated with or without the EGFR inhibitor (ZD1839, ZD), Src inhibitor (Dasatinib, Das), or the Stat3 inhibitor (S3I-201) for 1 or 24 h (B), or from nuclear extracts pre-incubated for 2 h with or without 100 µM pY1068, pY1086, or SPI peptide (C) and probing for EGFR, Src, Stat3; or (D) immunoblotting analysis of nuclear extracts prepared from Panc-1 cells treated or untreated with phenylarsine oxide (PAO) and probing for Src, Stat3, EGFR. Bands corresponding to proteins in gel are shown; input: except where indicated, represents the immunoblotting for the respective immunoprecipitated protein in the same amount of lysate or nuclear extract used in the assay; Data are representative of 3 independent studies.

    Article Snippet: Compared to anti-Stat3 or anti-Src antibody, the particle size increase upon addition of anti-EGFR antibody to the assay solution is much smaller, only slightly higher than the non-specific rabbit IgG.

    Techniques: Immunoprecipitation, Transfection, Incubation

    Immunofluorescence with laser-scanning confocal microscopy of EGFR, Src and Stat3 association in HPDEC or Panc-1 cells. Cultured normal human pancreatic duct epithelial cells (HPDEC) (A) or pancreatic cancer, Panc-1 cells (B) were fixed, stained with primary antibodies against EGFR, Src and Stat3 and their corresponding secondary antibodies, ALexaFLuor405 (goat anti-mouse, EGFR, red), AlexaFluor488 (donkey anti-rabbit, Src, blue) and AlexaFluor546 (goat anti-rat, Stat3, green) and analyzed by laser-scanning confocal microscopy for localization (single) and colocalization (merge) studies of EGFR (red), Src (blue) and Stat3 (green) and the effects of treatment (i) without or (ii) with ZD1839 (ZD) or (iii) Dasatinib (Das) for the indicated times. Confocal images were collected using Leica TCS SP5 microscopes; Cyan, magenta, yellow and white/pale yellow arrows denote merged colors; single, one color capture, merged, three-color capture. Data are representative of 3 independent studies.

    Journal: PLoS ONE

    Article Title: A Functional Nuclear Epidermal Growth Factor Receptor, Src and Stat3 Heteromeric Complex in Pancreatic Cancer Cells

    doi: 10.1371/journal.pone.0019605

    Figure Lengend Snippet: Immunofluorescence with laser-scanning confocal microscopy of EGFR, Src and Stat3 association in HPDEC or Panc-1 cells. Cultured normal human pancreatic duct epithelial cells (HPDEC) (A) or pancreatic cancer, Panc-1 cells (B) were fixed, stained with primary antibodies against EGFR, Src and Stat3 and their corresponding secondary antibodies, ALexaFLuor405 (goat anti-mouse, EGFR, red), AlexaFluor488 (donkey anti-rabbit, Src, blue) and AlexaFluor546 (goat anti-rat, Stat3, green) and analyzed by laser-scanning confocal microscopy for localization (single) and colocalization (merge) studies of EGFR (red), Src (blue) and Stat3 (green) and the effects of treatment (i) without or (ii) with ZD1839 (ZD) or (iii) Dasatinib (Das) for the indicated times. Confocal images were collected using Leica TCS SP5 microscopes; Cyan, magenta, yellow and white/pale yellow arrows denote merged colors; single, one color capture, merged, three-color capture. Data are representative of 3 independent studies.

    Article Snippet: Compared to anti-Stat3 or anti-Src antibody, the particle size increase upon addition of anti-EGFR antibody to the assay solution is much smaller, only slightly higher than the non-specific rabbit IgG.

    Techniques: Immunofluorescence, Confocal Microscopy, Cell Culture, Staining

    Co-immunoprecipitation with immunoblotting analysis of EGFR, Src and Stat3 complex in the nucleus and the sub-cellular distribution of EGFR, Src and Stat3. (A and B) Immunoblotting analyses of immunecomplexes of EGFR (IP:EGFR), Src (IP:Src), Stat3 (IP:Stat3), EGFR/Src (IP:EGFR/IP:Src), or of non-specific IgG non-immuneprecpitate prepared from nuclear extracts of Panc-1 or Colo-357 cells and probing for Stat3, EGFR, Src, or the Tata-binding protein (TBP); and (C), immunoblotting analysis of membrane (mem) and cytosolic (cyto) fractions and of nuclear (nuc) extracts from Panc-1 cells probing for (i) EGFR, (ii) Stat3 and (iii) Src. Bands corresponding to proteins in gel are shown; input: except where indicated, represents the immunoblotting for the respective immunoprecipitated protein in the same amount of nuclear extract used in the assay; IP:EGFR/IP:Src, sequential immunoprecipitation with anti-EGFR and then anti-Src antibody; Data are representative of 3 independent studies.

    Journal: PLoS ONE

    Article Title: A Functional Nuclear Epidermal Growth Factor Receptor, Src and Stat3 Heteromeric Complex in Pancreatic Cancer Cells

    doi: 10.1371/journal.pone.0019605

    Figure Lengend Snippet: Co-immunoprecipitation with immunoblotting analysis of EGFR, Src and Stat3 complex in the nucleus and the sub-cellular distribution of EGFR, Src and Stat3. (A and B) Immunoblotting analyses of immunecomplexes of EGFR (IP:EGFR), Src (IP:Src), Stat3 (IP:Stat3), EGFR/Src (IP:EGFR/IP:Src), or of non-specific IgG non-immuneprecpitate prepared from nuclear extracts of Panc-1 or Colo-357 cells and probing for Stat3, EGFR, Src, or the Tata-binding protein (TBP); and (C), immunoblotting analysis of membrane (mem) and cytosolic (cyto) fractions and of nuclear (nuc) extracts from Panc-1 cells probing for (i) EGFR, (ii) Stat3 and (iii) Src. Bands corresponding to proteins in gel are shown; input: except where indicated, represents the immunoblotting for the respective immunoprecipitated protein in the same amount of nuclear extract used in the assay; IP:EGFR/IP:Src, sequential immunoprecipitation with anti-EGFR and then anti-Src antibody; Data are representative of 3 independent studies.

    Article Snippet: Compared to anti-Stat3 or anti-Src antibody, the particle size increase upon addition of anti-EGFR antibody to the assay solution is much smaller, only slightly higher than the non-specific rabbit IgG.

    Techniques: Immunoprecipitation, Binding Assay

    Studies of protein complex and protein binding partners using the Detection and Analysis through Nanoparticle Sizing technology. (A) Kinetic binding assay of EGFR-gold nanoparticle (GNP) probe (or mouse IgG1-GNP probe as negative control) binding to (i) EGFR protein and its complex from Panc-1 nuclear extracts, and the (ii) inhibitory effect of the mouse monoclonal anti-EGFR antibody on the EGFR-GNP probe binding to the EGFR protein; and (B) Protein complex binding partner analysis whereby the polyclonal anti-Stat3, anti-Src or anti-EGFR antibody or the non-specific rabbit IgG (negative control) is added to the assay solution prepared from the (i) non-specific mouse IgG1-GNP probe (negative control), or (ii) anti-EGFR-GNP probe; Data are representative of 4 independent studies.

    Journal: PLoS ONE

    Article Title: A Functional Nuclear Epidermal Growth Factor Receptor, Src and Stat3 Heteromeric Complex in Pancreatic Cancer Cells

    doi: 10.1371/journal.pone.0019605

    Figure Lengend Snippet: Studies of protein complex and protein binding partners using the Detection and Analysis through Nanoparticle Sizing technology. (A) Kinetic binding assay of EGFR-gold nanoparticle (GNP) probe (or mouse IgG1-GNP probe as negative control) binding to (i) EGFR protein and its complex from Panc-1 nuclear extracts, and the (ii) inhibitory effect of the mouse monoclonal anti-EGFR antibody on the EGFR-GNP probe binding to the EGFR protein; and (B) Protein complex binding partner analysis whereby the polyclonal anti-Stat3, anti-Src or anti-EGFR antibody or the non-specific rabbit IgG (negative control) is added to the assay solution prepared from the (i) non-specific mouse IgG1-GNP probe (negative control), or (ii) anti-EGFR-GNP probe; Data are representative of 4 independent studies.

    Article Snippet: Compared to anti-Stat3 or anti-Src antibody, the particle size increase upon addition of anti-EGFR antibody to the assay solution is much smaller, only slightly higher than the non-specific rabbit IgG.

    Techniques: Protein Binding, Binding Assay, Negative Control

    Co-immunoprecipitation with immunoblotting analysis of EGFR, Src and Stat3 association in Panc-1 and Colo-357 cells. Immunoblotting analyses of immunecomplexes of EGFR (IP:EGFR), Src (IP:Src), and Stat3 (IP:Stat3), or of non-specific IgG non-immunoprecipitate prepared from whole-cell lysates of Panc-1 or Colo-357 cells untransfected (A and B) or transfected with EGFR siRNA, Src siRNA, or control (con) siRNA (C) and probing for Src, Stat3 and EGFR in the absence (A and C) or presence (B) of Stat3 blocking peptide (Stat3 BP), Src blocking peptide (Src BP) or EGFR blocking peptide (EGFR BP). Bands corresponding to proteins in gel are shown; input: except where indicated, represents the immunoblotting for the respective immunoprecipitated protein in the same amount of lysate used in the assay; Data are representative of 3 independent studies.

    Journal: PLoS ONE

    Article Title: A Functional Nuclear Epidermal Growth Factor Receptor, Src and Stat3 Heteromeric Complex in Pancreatic Cancer Cells

    doi: 10.1371/journal.pone.0019605

    Figure Lengend Snippet: Co-immunoprecipitation with immunoblotting analysis of EGFR, Src and Stat3 association in Panc-1 and Colo-357 cells. Immunoblotting analyses of immunecomplexes of EGFR (IP:EGFR), Src (IP:Src), and Stat3 (IP:Stat3), or of non-specific IgG non-immunoprecipitate prepared from whole-cell lysates of Panc-1 or Colo-357 cells untransfected (A and B) or transfected with EGFR siRNA, Src siRNA, or control (con) siRNA (C) and probing for Src, Stat3 and EGFR in the absence (A and C) or presence (B) of Stat3 blocking peptide (Stat3 BP), Src blocking peptide (Src BP) or EGFR blocking peptide (EGFR BP). Bands corresponding to proteins in gel are shown; input: except where indicated, represents the immunoblotting for the respective immunoprecipitated protein in the same amount of lysate used in the assay; Data are representative of 3 independent studies.

    Article Snippet: Compared to anti-Stat3 or anti-Src antibody, the particle size increase upon addition of anti-EGFR antibody to the assay solution is much smaller, only slightly higher than the non-specific rabbit IgG.

    Techniques: Immunoprecipitation, Transfection, Blocking Assay

    Exosome-treated Caco-2 cells showed reduced PTEN and increased phosphor-Akt amounts. A , B , Western blot was used to assess the expression levels of p-EGFR, EGFR, PTEN p-Akt, Akt and GAPDH in RKO, Caco-2 cells and their derived exosomes. Data are reported as means±SD. *P

    Journal: Brazilian Journal of Medical and Biological Research

    Article Title: Exosomes promote cetuximab resistance via the PTEN/Akt pathway in colon cancer cells

    doi: 10.1590/1414-431X20176472

    Figure Lengend Snippet: Exosome-treated Caco-2 cells showed reduced PTEN and increased phosphor-Akt amounts. A , B , Western blot was used to assess the expression levels of p-EGFR, EGFR, PTEN p-Akt, Akt and GAPDH in RKO, Caco-2 cells and their derived exosomes. Data are reported as means±SD. *P

    Article Snippet: Antibodies targeting EGFR, phosphor-EGFR (Tyr1068), Akt, phosphor-Akt (Ser473), PTEN, PI3-kinase and calreticulin were obtained from Cell Signaling Technology (USA).

    Techniques: Western Blot, Expressing, Derivative Assay

    RKO-derived exosomes induced cetuximab resistance in Caco-2 cells via the PTEN/Akt pathway. A , Western blot was used to detect the expression levels of p-EGFR, EGFR, p-Akt, Akt, PTEN, and GAPDH in Caco-2 cells treated with cetuximab (10 μg/mL) for 48 h after pre-treatment with RKO-derived exosomes (50 μg/mL) for 48 h. B , The MTT assay showed that the Akt inhibitor LY294002 reversed the RKO-derived exosome induced C225 drug resistance in Caco-2 cells. Data are reported as means±SD. *P

    Journal: Brazilian Journal of Medical and Biological Research

    Article Title: Exosomes promote cetuximab resistance via the PTEN/Akt pathway in colon cancer cells

    doi: 10.1590/1414-431X20176472

    Figure Lengend Snippet: RKO-derived exosomes induced cetuximab resistance in Caco-2 cells via the PTEN/Akt pathway. A , Western blot was used to detect the expression levels of p-EGFR, EGFR, p-Akt, Akt, PTEN, and GAPDH in Caco-2 cells treated with cetuximab (10 μg/mL) for 48 h after pre-treatment with RKO-derived exosomes (50 μg/mL) for 48 h. B , The MTT assay showed that the Akt inhibitor LY294002 reversed the RKO-derived exosome induced C225 drug resistance in Caco-2 cells. Data are reported as means±SD. *P

    Article Snippet: Antibodies targeting EGFR, phosphor-EGFR (Tyr1068), Akt, phosphor-Akt (Ser473), PTEN, PI3-kinase and calreticulin were obtained from Cell Signaling Technology (USA).

    Techniques: Derivative Assay, Western Blot, Expressing, MTT Assay

    EGFR activation and Erlotinib antitumor activity in LCSC-derived ADC xenografts. ( a ) Immunoblot analysis of EGFR tyr1068 in sensitive (LCSC5) or resistant (LCSC7) LCSCs and in the corresponding xenografts untreated (−) or treated (+) with erlotinib. ( b ) Growth curves of the same control or erlotinib-treated xenografts as in ( a ). Mean±S.D. of three independent experiments is shown. *** P

    Journal: Cell Death & Disease

    Article Title: Tyr1068-phosphorylated epidermal growth factor receptor (EGFR) predicts cancer stem cell targeting by erlotinib in preclinical models of wild-type EGFR lung cancer

    doi: 10.1038/cddis.2015.217

    Figure Lengend Snippet: EGFR activation and Erlotinib antitumor activity in LCSC-derived ADC xenografts. ( a ) Immunoblot analysis of EGFR tyr1068 in sensitive (LCSC5) or resistant (LCSC7) LCSCs and in the corresponding xenografts untreated (−) or treated (+) with erlotinib. ( b ) Growth curves of the same control or erlotinib-treated xenografts as in ( a ). Mean±S.D. of three independent experiments is shown. *** P

    Article Snippet: Rabbit polyclonal anti-Phospho-EGFR (Tyr1068), -Phospho-EGFR (tyr1173), -Phospho-Akt (Ser473) -Akt, -Caspase-3 (8G10) and -Phospho-Stat3 (Ser727) and mouse monoclonal anti-STAT3 were purchased from Cell Signaling (Beverly, MA, USA).

    Techniques: Activation Assay, Activity Assay, Derivative Assay