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Santa Cruz Biotechnology egfr
Involvement of NEU3 in epidermal growth factor (EGF)‐induced elevation of MMP9 expression in head and neck squamous cell carcinoma cells. (a) Attenuation of EGF‐induced MMP9 elevation in NEU3‐silenced HSC‐2 and SAS cells in terms of mRNA levels. (b) Attenuation of EGF‐induced MMP2 and MMP9 secretion and activity by NEU3 silencing in HSC‐2 cells, as assessed by gelatin zymography. (c) Enhanced phosphorylation of EGF receptor <t>(EGFR),</t> <t>ERK,</t> and Akt by NEU3 overexpression and its reduction by NEU3 silencing, as assessed by Western blotting. Each value shown under the blot represents a value relative to that in the vector control (Vec) without EGF. (d) Abrogation of NEU3‐mediated as well as EGF‐induced augmentation of MMP9 expression by EGFR inhibitor AG1478.
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1) Product Images from "Upregulation of sialidase NEU3 in head and neck squamous cell carcinoma associated with lymph node metastasis"

Article Title: Upregulation of sialidase NEU3 in head and neck squamous cell carcinoma associated with lymph node metastasis

Journal: Cancer Science

doi: 10.1111/cas.12810

Involvement of NEU3 in epidermal growth factor (EGF)‐induced elevation of MMP9 expression in head and neck squamous cell carcinoma cells. (a) Attenuation of EGF‐induced MMP9 elevation in NEU3‐silenced HSC‐2 and SAS cells in terms of mRNA levels. (b) Attenuation of EGF‐induced MMP2 and MMP9 secretion and activity by NEU3 silencing in HSC‐2 cells, as assessed by gelatin zymography. (c) Enhanced phosphorylation of EGF receptor (EGFR), ERK, and Akt by NEU3 overexpression and its reduction by NEU3 silencing, as assessed by Western blotting. Each value shown under the blot represents a value relative to that in the vector control (Vec) without EGF. (d) Abrogation of NEU3‐mediated as well as EGF‐induced augmentation of MMP9 expression by EGFR inhibitor AG1478.
Figure Legend Snippet: Involvement of NEU3 in epidermal growth factor (EGF)‐induced elevation of MMP9 expression in head and neck squamous cell carcinoma cells. (a) Attenuation of EGF‐induced MMP9 elevation in NEU3‐silenced HSC‐2 and SAS cells in terms of mRNA levels. (b) Attenuation of EGF‐induced MMP2 and MMP9 secretion and activity by NEU3 silencing in HSC‐2 cells, as assessed by gelatin zymography. (c) Enhanced phosphorylation of EGF receptor (EGFR), ERK, and Akt by NEU3 overexpression and its reduction by NEU3 silencing, as assessed by Western blotting. Each value shown under the blot represents a value relative to that in the vector control (Vec) without EGF. (d) Abrogation of NEU3‐mediated as well as EGF‐induced augmentation of MMP9 expression by EGFR inhibitor AG1478.

Techniques Used: Expressing, Activity Assay, Zymography, Over Expression, Western Blot, Plasmid Preparation

2) Product Images from "Neurotensin stimulates expression of early growth response gene-1 and EGF receptor through MAP kinase activation in human colonic epithelial cells"

Article Title: Neurotensin stimulates expression of early growth response gene-1 and EGF receptor through MAP kinase activation in human colonic epithelial cells

Journal:

doi: 10.1002/ijc.22407

Effect of Egr-1 siRNA on NT-stimulated EGFR promoter activity. ( a ) Hela cells were transiently transfected with either Egr-1 siRNA or its control siRNA as described in the text. Equal amount of cell proteins were used to determine the expression of endogenous
Figure Legend Snippet: Effect of Egr-1 siRNA on NT-stimulated EGFR promoter activity. ( a ) Hela cells were transiently transfected with either Egr-1 siRNA or its control siRNA as described in the text. Equal amount of cell proteins were used to determine the expression of endogenous

Techniques Used: Activity Assay, Transfection, Expressing

3) Product Images from "Augmentation of radiation response by panitumumab in models of upper aerodigestive tract cancer"

Article Title: Augmentation of radiation response by panitumumab in models of upper aerodigestive tract cancer

Journal:

doi: 10.1016/j.ijrobp.2008.06.1490

Panitumumab blocks radiation-induced EGFR nuclear translocation in SCC-1 cells. Panitumumab (PNT, 25 nM) or non-specific IgG was adminstered 1 hour before radiation (3 Gy). (A) Cells were lysed 20 minutes after radiation and cytosolic and nuclear fractions
Figure Legend Snippet: Panitumumab blocks radiation-induced EGFR nuclear translocation in SCC-1 cells. Panitumumab (PNT, 25 nM) or non-specific IgG was adminstered 1 hour before radiation (3 Gy). (A) Cells were lysed 20 minutes after radiation and cytosolic and nuclear fractions

Techniques Used: Translocation Assay

4) Product Images from "Nuclear EGFR Contributes to Acquired Resistance to Cetuximab"

Article Title: Nuclear EGFR Contributes to Acquired Resistance to Cetuximab

Journal: Oncogene

doi: 10.1038/onc.2009.234

Overexpression of a NLS tagged EGFR in cetuximab-resistant cells confers resistance to cetuximab in vivo Male athymic nude mice were injected subcutaneously with 1×10 6 cetuximab-sensitive parental cells (HP) or CMV-EGFR-NLS/Myc clone cells (Vector only, Clone 4, Clone 5 and Clone 10) into the dorsal flank. Once tumors reached a volume 120-180mm 3 mice were treated with 0.1 mg IgG or cetuximab twice weekly. Tumor diameters were measured serially with calipers and tumor volumes were calculated. Points, mean tumor volume of eight mice per group; bars, SD. T -test was used to compare tumor volumes between cetuximab treated and control IgG mice. *, P
Figure Legend Snippet: Overexpression of a NLS tagged EGFR in cetuximab-resistant cells confers resistance to cetuximab in vivo Male athymic nude mice were injected subcutaneously with 1×10 6 cetuximab-sensitive parental cells (HP) or CMV-EGFR-NLS/Myc clone cells (Vector only, Clone 4, Clone 5 and Clone 10) into the dorsal flank. Once tumors reached a volume 120-180mm 3 mice were treated with 0.1 mg IgG or cetuximab twice weekly. Tumor diameters were measured serially with calipers and tumor volumes were calculated. Points, mean tumor volume of eight mice per group; bars, SD. T -test was used to compare tumor volumes between cetuximab treated and control IgG mice. *, P

Techniques Used: Over Expression, In Vivo, Mouse Assay, Injection, Plasmid Preparation

Potential mechanism for resistance to cetuximab A ) Cetuximab-sensitive cells depend on classical EGFR membrane signaling. B ) Tumor cells that acquire resistance to cetuximab gain nEGFR as a second compartment of proliferation. C ) Cetuximab can abrogate signals from plasma membrane EGFR but not nEGFR; nEGFR continues to send proliferative signals by modulation of Cyclin D1, B-myb, Aurora kinase K and regulation of PCNA. D ) The SFK inhibitor dasatinib inhibits nuclear translocation of the EGFR from the plasma membrane leading to increased EGFR on the plasma membrane and restoring sensitivity to cetuximab.
Figure Legend Snippet: Potential mechanism for resistance to cetuximab A ) Cetuximab-sensitive cells depend on classical EGFR membrane signaling. B ) Tumor cells that acquire resistance to cetuximab gain nEGFR as a second compartment of proliferation. C ) Cetuximab can abrogate signals from plasma membrane EGFR but not nEGFR; nEGFR continues to send proliferative signals by modulation of Cyclin D1, B-myb, Aurora kinase K and regulation of PCNA. D ) The SFK inhibitor dasatinib inhibits nuclear translocation of the EGFR from the plasma membrane leading to increased EGFR on the plasma membrane and restoring sensitivity to cetuximab.

Techniques Used: Translocation Assay

Src family kinases mediate ligand-induced EGFR translocation to the nucleus A ) Dasatinib inhibits HER family ligands signaling in parental cells (HP). HP cells were untreated, treated for 24 hours with 50 nM of dasatinib alone, or followed by 200 ng/ml of indicated ligand for 1 hour prior to harvesting. Nuclear protein was collected and fractionated by SDS-PAGE followed by immunoblotting for EGFR. histone H3 was used as loading control. B ) Dasatinib inhibits nuclear expression of EGFR in cetuximab-resistant cell lines. Parental cells (HP) and cetuximab-resistant cell lines (HC1, HC4, HC8) were treated with 50 nM of dasatinib for 24 hours. After cells were harvested, cytoplasmic and nuclear protein was fractionated by SDS-PAGE followed by immunoblotting for EGFR. α-tubulin and histone H3 were used as loading controls and purity controls of each cellular fraction. Expression of nEGFR after dasatinib treatment in cetuximab-resistant clones was quantitated using ImageJ software and normalized against the amounts of untreated cells. C ) Dasatinib treatment lead to increased membrane-bound EGFR in cetuximab-resistant cells by flow cytometry analysis. Parental cells (HP) and cetuximab-resistant cells (HC1, HC4 and HC8) were treated with DMSO or 50 nM of dasatinib for 24 hours and membrane expression is represented relative to untreated controls. Mean surface expression of EGFR is represented +/- SEM (n=3). Flow cytometric plots of representative experiments are presented. Shaded histograms represent dasatinib treatment. Controls (dotted line) represent cells labeled with FITC-conjugated normal mouse IgG *, P
Figure Legend Snippet: Src family kinases mediate ligand-induced EGFR translocation to the nucleus A ) Dasatinib inhibits HER family ligands signaling in parental cells (HP). HP cells were untreated, treated for 24 hours with 50 nM of dasatinib alone, or followed by 200 ng/ml of indicated ligand for 1 hour prior to harvesting. Nuclear protein was collected and fractionated by SDS-PAGE followed by immunoblotting for EGFR. histone H3 was used as loading control. B ) Dasatinib inhibits nuclear expression of EGFR in cetuximab-resistant cell lines. Parental cells (HP) and cetuximab-resistant cell lines (HC1, HC4, HC8) were treated with 50 nM of dasatinib for 24 hours. After cells were harvested, cytoplasmic and nuclear protein was fractionated by SDS-PAGE followed by immunoblotting for EGFR. α-tubulin and histone H3 were used as loading controls and purity controls of each cellular fraction. Expression of nEGFR after dasatinib treatment in cetuximab-resistant clones was quantitated using ImageJ software and normalized against the amounts of untreated cells. C ) Dasatinib treatment lead to increased membrane-bound EGFR in cetuximab-resistant cells by flow cytometry analysis. Parental cells (HP) and cetuximab-resistant cells (HC1, HC4 and HC8) were treated with DMSO or 50 nM of dasatinib for 24 hours and membrane expression is represented relative to untreated controls. Mean surface expression of EGFR is represented +/- SEM (n=3). Flow cytometric plots of representative experiments are presented. Shaded histograms represent dasatinib treatment. Controls (dotted line) represent cells labeled with FITC-conjugated normal mouse IgG *, P

Techniques Used: Translocation Assay, SDS Page, Expressing, Clone Assay, Software, Flow Cytometry, Cytometry, Labeling

EGFR tagged with nuclear localization sequence confers resistance to cetuximab in vitro A ) A schematic representation of the CMV-EGFR-NLS/Myc construct is shown. EGFR-NLS/Myc was driven by the CMV promoter. The cetuximab-sensitive NSCLC line NCI-H226 was infected with indicated constructs. Represented is three individual clones and vector control (V 0 ; vector only, C 4 , C 5 and C 10 ). Cytoplasmic and nuclear protein from each clone was collected and immunoprecipitated with an anti-myc antibody, fractionated on SDS-PAGE and immunoblotted with the indicated antibodies. α-tubulin and histone H3 were used as loading and purity control for cytosolic and nuclear fractions, respectively. Immunofluorescence of nEGFR staining in CMV-EGFR-NLS/Myc clones. EGFR (green), DNA (blue), stained by PI. V 0 ; vector clone, C 4 , C 5 and C 10 ; CMV-EGFR-NLS/Myc clones. 400× magnification. cEGFR; cytoplasmic EGFR, nEGFR; nuclear EGFR. B ) CMV-EGFR-NLS/Myc expressed in NCI-H226 leads to increased cyclin D1 and B-myb expression. Nuclear protein from EGFR-NLS/myc clones was collected and fractionated by SDS-PAGE followed by immunoblotting for the indicated proteins. histone H3 was used as a loading control. Expression of cyclin D1 and B-myb in CMV-EGFR-NLS/Myc clones (C 4 , C 5 and C 10 ) were quantitated using ImageJ software and normalized against the amounts of those proteins in vector control (V 0 ). V 0 ; vector clone, C 4 , C 5 and C 10 ; CMV-EGFR-NLS/Myc clones. C ) Growth response to cetuximab of three individual clones and vector control (V 0 ; vector only, C 4 , C 5 and C 10 ). CMV-EGFR-NLS/Myc-tag clones (C 4 , C 5 and C 10 ) were treated with 100 nM of cetuximab and growth was measured using the growth proliferation assay and plotted as growth relative to untreated control. Data points are represented as mean +/- SEM. (n=3). *, P
Figure Legend Snippet: EGFR tagged with nuclear localization sequence confers resistance to cetuximab in vitro A ) A schematic representation of the CMV-EGFR-NLS/Myc construct is shown. EGFR-NLS/Myc was driven by the CMV promoter. The cetuximab-sensitive NSCLC line NCI-H226 was infected with indicated constructs. Represented is three individual clones and vector control (V 0 ; vector only, C 4 , C 5 and C 10 ). Cytoplasmic and nuclear protein from each clone was collected and immunoprecipitated with an anti-myc antibody, fractionated on SDS-PAGE and immunoblotted with the indicated antibodies. α-tubulin and histone H3 were used as loading and purity control for cytosolic and nuclear fractions, respectively. Immunofluorescence of nEGFR staining in CMV-EGFR-NLS/Myc clones. EGFR (green), DNA (blue), stained by PI. V 0 ; vector clone, C 4 , C 5 and C 10 ; CMV-EGFR-NLS/Myc clones. 400× magnification. cEGFR; cytoplasmic EGFR, nEGFR; nuclear EGFR. B ) CMV-EGFR-NLS/Myc expressed in NCI-H226 leads to increased cyclin D1 and B-myb expression. Nuclear protein from EGFR-NLS/myc clones was collected and fractionated by SDS-PAGE followed by immunoblotting for the indicated proteins. histone H3 was used as a loading control. Expression of cyclin D1 and B-myb in CMV-EGFR-NLS/Myc clones (C 4 , C 5 and C 10 ) were quantitated using ImageJ software and normalized against the amounts of those proteins in vector control (V 0 ). V 0 ; vector clone, C 4 , C 5 and C 10 ; CMV-EGFR-NLS/Myc clones. C ) Growth response to cetuximab of three individual clones and vector control (V 0 ; vector only, C 4 , C 5 and C 10 ). CMV-EGFR-NLS/Myc-tag clones (C 4 , C 5 and C 10 ) were treated with 100 nM of cetuximab and growth was measured using the growth proliferation assay and plotted as growth relative to untreated control. Data points are represented as mean +/- SEM. (n=3). *, P

Techniques Used: Sequencing, In Vitro, Construct, Infection, Clone Assay, Plasmid Preparation, Immunoprecipitation, SDS Page, Immunofluorescence, Staining, Expressing, Software, Proliferation Assay

5) Product Images from "Necrotic renal epithelial cell inhibits renal interstitial fibroblast activation: role of protein tyrosine phosphatase 1B"

Article Title: Necrotic renal epithelial cell inhibits renal interstitial fibroblast activation: role of protein tyrosine phosphatase 1B

Journal: American Journal of Physiology - Renal Physiology

doi: 10.1152/ajprenal.00564.2012

Necrotic RPTC induce PTP1B tyrosine phosphorylaton and its interaction with EGFR. NRK-49F cells were treated with RPTC-Sup for 36 h and then cell lysates were subjected to immunoblotting for PTP1B and GAPDH ( A ). Normally cultured NRK-49F cells were treated
Figure Legend Snippet: Necrotic RPTC induce PTP1B tyrosine phosphorylaton and its interaction with EGFR. NRK-49F cells were treated with RPTC-Sup for 36 h and then cell lysates were subjected to immunoblotting for PTP1B and GAPDH ( A ). Normally cultured NRK-49F cells were treated

Techniques Used: Cell Culture

6) Product Images from "USP8 is a novel target for overcoming gefitinib-resistance in lung cancer"

Article Title: USP8 is a novel target for overcoming gefitinib-resistance in lung cancer

Journal: Clinical cancer research : an official journal of the American Association for Cancer Research

doi: 10.1158/1078-0432.CCR-12-3696

Inhibition of USP8 enhances co-localization between ubiquitin and target receptor tyrosine kinases. H1650 cells were treated with 1 µM USP8i for 90 min and then immunostained to detect (A) EGFR or (B) ErbB2 (green) and ubiquitin (red). The merged
Figure Legend Snippet: Inhibition of USP8 enhances co-localization between ubiquitin and target receptor tyrosine kinases. H1650 cells were treated with 1 µM USP8i for 90 min and then immunostained to detect (A) EGFR or (B) ErbB2 (green) and ubiquitin (red). The merged

Techniques Used: Inhibition

7) Product Images from "FIH-1 Disrupts an LRRK1/EGFR Complex to Positively Regulate Keratinocyte Migration"

Article Title: FIH-1 Disrupts an LRRK1/EGFR Complex to Positively Regulate Keratinocyte Migration

Journal: The American Journal of Pathology

doi: 10.1016/j.ajpath.2014.08.014

FIH-1 enhances EGFR signaling to promote keratinocyte cell migration. HEKs ( A ) and HCEKs ( B ) were retrovirally transduced with either FIH-1 or empty vector (LZRS). Protein lysates from HEKs or HCEKs were harvested for immunoprecipitation
Figure Legend Snippet: FIH-1 enhances EGFR signaling to promote keratinocyte cell migration. HEKs ( A ) and HCEKs ( B ) were retrovirally transduced with either FIH-1 or empty vector (LZRS). Protein lysates from HEKs or HCEKs were harvested for immunoprecipitation

Techniques Used: Migration, Transduction, Plasmid Preparation, Immunoprecipitation

FIH-1 forms a complex with LRRK1 that affects EGFR activation and keratinocyte migration. A and B: Interaction of LRRK1 with FIH-1. Complex formation of ectopically overexpressed ( A ) or endogenous ( B ) FIH-1 with endogenous LRRK1 in HEKs was detected by
Figure Legend Snippet: FIH-1 forms a complex with LRRK1 that affects EGFR activation and keratinocyte migration. A and B: Interaction of LRRK1 with FIH-1. Complex formation of ectopically overexpressed ( A ) or endogenous ( B ) FIH-1 with endogenous LRRK1 in HEKs was detected by

Techniques Used: Activation Assay, Migration

FIH-1/LRRK1 regulates EGFR signaling in part by interfering with endosomal trafficking. HEKs ( A and B ) and limbal-derived corneal epithelial hTCEpi cells ( C and D ) were transfected with siLRRK1 ( A and C ), siFIH-1 ( B and D ), or siCTRL. After transfection
Figure Legend Snippet: FIH-1/LRRK1 regulates EGFR signaling in part by interfering with endosomal trafficking. HEKs ( A and B ) and limbal-derived corneal epithelial hTCEpi cells ( C and D ) were transfected with siLRRK1 ( A and C ), siFIH-1 ( B and D ), or siCTRL. After transfection

Techniques Used: Derivative Assay, Transfection

8) Product Images from "Nuclear Epidermal Growth Factor Receptor is a Functional Molecular Target in Triple-Negative Breast Cancer"

Article Title: Nuclear Epidermal Growth Factor Receptor is a Functional Molecular Target in Triple-Negative Breast Cancer

Journal: Molecular cancer therapeutics

doi: 10.1158/1535-7163.MCT-13-1021

Src Family Kinases mediate nEGFR translocation in TNBC (A) Constitutively active Src (caSrc) enhances nEGFR translocation in TNBC cell lines . Cells were transfected with caSrc or an empty vector control for 48 hr prior to stimulation with EGF (5 nM, 45 min) to induce nEGFR translocation. Non-nuclear and nuclear proteins were harvested. nEGFR expression was quantitated using ImageJ software (B) A negative regulator of Src, src-like adaptor protein (SLAP), blocks nEGFR translocation in TNBC cell lines. Cells were transfected with SLAP-FLAG or an empty vector control for 48 hr prior to harvesting non-nuclear and nuclear proteins. nEGFR expression was analyzed. (C) SLAP can interact with EGFR and decrease EGFR activation at Tyrosine 1101. Cells were transfected with SLAP-FLAG or an empty vector control for 48 hr prior to harvesting whole cell lysate. 250 ug of cell lysate was immunoprecipitated with an anti-SLAP antibody. The same lysate was subjected to immunoblot analysis for activation of EGFR at Tyrosine 1101. pEGFR-Y1101 activity was quantitated using ImageJ software. Inset 1: EGFR mutated at Tyrosine 1101 is deficient in nuclear localization. Vector, EGFR-WT, and EGFR-Y1101F were transfected into CHOK1 cells for 48 hr prior to stimulation with EGF (5 nM, 45 min). Non-nuclear and nuclear proteins were harvested, and nEGFR expression was analyzed.
Figure Legend Snippet: Src Family Kinases mediate nEGFR translocation in TNBC (A) Constitutively active Src (caSrc) enhances nEGFR translocation in TNBC cell lines . Cells were transfected with caSrc or an empty vector control for 48 hr prior to stimulation with EGF (5 nM, 45 min) to induce nEGFR translocation. Non-nuclear and nuclear proteins were harvested. nEGFR expression was quantitated using ImageJ software (B) A negative regulator of Src, src-like adaptor protein (SLAP), blocks nEGFR translocation in TNBC cell lines. Cells were transfected with SLAP-FLAG or an empty vector control for 48 hr prior to harvesting non-nuclear and nuclear proteins. nEGFR expression was analyzed. (C) SLAP can interact with EGFR and decrease EGFR activation at Tyrosine 1101. Cells were transfected with SLAP-FLAG or an empty vector control for 48 hr prior to harvesting whole cell lysate. 250 ug of cell lysate was immunoprecipitated with an anti-SLAP antibody. The same lysate was subjected to immunoblot analysis for activation of EGFR at Tyrosine 1101. pEGFR-Y1101 activity was quantitated using ImageJ software. Inset 1: EGFR mutated at Tyrosine 1101 is deficient in nuclear localization. Vector, EGFR-WT, and EGFR-Y1101F were transfected into CHOK1 cells for 48 hr prior to stimulation with EGF (5 nM, 45 min). Non-nuclear and nuclear proteins were harvested, and nEGFR expression was analyzed.

Techniques Used: Translocation Assay, Transfection, Plasmid Preparation, Expressing, Software, Activation Assay, Immunoprecipitation, Activity Assay

TNBC cell lines and human tumors express nuclear localized EGFR (A) TNBC cells express total and activated forms of EGFR. Whole cell lysate was harvested from six TNBC cell lines and two HER2 positive cell lines. α-Tubulin was used as a loading control. (B) TNBC cells express nEGFR. Cell lines were harvested for nuclear proteins. α-Tubulin, calnexin, and Histone H3 were used as loading and purity controls, respectively. Confocal IF microscopy depicts nEGFR expression. Merged images were magnified to depict nEGFR (Confocal zoom, white arrows). A single Z-Slice image depicts overlap between blue and red signal (white dashed-line boxes). Magnification 600X. EGFR primary antibody specificity was validated with siEGFR and blocking peptides. (C) Immunogold labeling of nEGFR. TNBC cells were fixed and processed for transmission EM. CY, cytoplasm; NE, nuclear envelope; NUC, nucleus; NOS, nucleolus. Images were digitally zoomed to highlight gold particles in the nucleus (black arrows). (D) Human TNBC tumors express nEGFR. IHC staining for EGFR was performed on a total of 74 TNBC patient tumor sections. Representative cases demonstrating nEGFR expression are depicted (black arrows).
Figure Legend Snippet: TNBC cell lines and human tumors express nuclear localized EGFR (A) TNBC cells express total and activated forms of EGFR. Whole cell lysate was harvested from six TNBC cell lines and two HER2 positive cell lines. α-Tubulin was used as a loading control. (B) TNBC cells express nEGFR. Cell lines were harvested for nuclear proteins. α-Tubulin, calnexin, and Histone H3 were used as loading and purity controls, respectively. Confocal IF microscopy depicts nEGFR expression. Merged images were magnified to depict nEGFR (Confocal zoom, white arrows). A single Z-Slice image depicts overlap between blue and red signal (white dashed-line boxes). Magnification 600X. EGFR primary antibody specificity was validated with siEGFR and blocking peptides. (C) Immunogold labeling of nEGFR. TNBC cells were fixed and processed for transmission EM. CY, cytoplasm; NE, nuclear envelope; NUC, nucleus; NOS, nucleolus. Images were digitally zoomed to highlight gold particles in the nucleus (black arrows). (D) Human TNBC tumors express nEGFR. IHC staining for EGFR was performed on a total of 74 TNBC patient tumor sections. Representative cases demonstrating nEGFR expression are depicted (black arrows).

Techniques Used: Microscopy, Expressing, Blocking Assay, Labeling, Transmission Assay, Immunohistochemistry, Staining

9) Product Images from "Thymosin beta 4 up-regulates miR-200a expression and induces differentiation and survival of rat brain progenitor cells"

Article Title: Thymosin beta 4 up-regulates miR-200a expression and induces differentiation and survival of rat brain progenitor cells

Journal: Journal of neurochemistry

doi: 10.1111/jnc.13394

Tβ4 treatment induces EGFR phosphorylation/activation and the expression of MBP, Mig-6 and miR-200
Figure Legend Snippet: Tβ4 treatment induces EGFR phosphorylation/activation and the expression of MBP, Mig-6 and miR-200

Techniques Used: Activation Assay, Expressing

Transfection with miR-200b-a-429 cluster enhances EGFR signaling and MBP synthesis, and inhibits Grb2 synthesis and ERK1phosphorylation
Figure Legend Snippet: Transfection with miR-200b-a-429 cluster enhances EGFR signaling and MBP synthesis, and inhibits Grb2 synthesis and ERK1phosphorylation

Techniques Used: Transfection

10) Product Images from "The mitogen-activated protein kinase kinase 4 (MKK4) has a pro-oncogenic role in skin cancer"

Article Title: The mitogen-activated protein kinase kinase 4 (MKK4) has a pro-oncogenic role in skin cancer

Journal: Cancer research

doi: 10.1158/0008-5472.CAN-09-3669

The loss of MKK4 prevents the phosphorylation of c-Jun in the skin treated with DMBA/TPA. mkk4skin +/+ and mkk4skin −/− animals were treated with DMBA/TPA. Controls correspond to age matched untreated mkk4skin +/+ mice. Sections of skin biopsies and tumors 20 weeks after TPA treatment were immunostained with antibodies to c-Jun, phospho (P)-c-Jun at Ser 73, EGFR, and p53. The results show that MKK4 is required for increased expression and phosphorylation of c-Jun, and increased expression of the c-Jun target EGFR. Elevated expression of p53 in MKK4-deficient epidermis of animals treated with DMBA/TPA is consistent with evidence that p53 is a tumor suppressor whose transcription can be repressed by c-Jun. Scale bar represents 20 μM.
Figure Legend Snippet: The loss of MKK4 prevents the phosphorylation of c-Jun in the skin treated with DMBA/TPA. mkk4skin +/+ and mkk4skin −/− animals were treated with DMBA/TPA. Controls correspond to age matched untreated mkk4skin +/+ mice. Sections of skin biopsies and tumors 20 weeks after TPA treatment were immunostained with antibodies to c-Jun, phospho (P)-c-Jun at Ser 73, EGFR, and p53. The results show that MKK4 is required for increased expression and phosphorylation of c-Jun, and increased expression of the c-Jun target EGFR. Elevated expression of p53 in MKK4-deficient epidermis of animals treated with DMBA/TPA is consistent with evidence that p53 is a tumor suppressor whose transcription can be repressed by c-Jun. Scale bar represents 20 μM.

Techniques Used: Mouse Assay, Expressing

11) Product Images from "Farnesoid X Receptor inhibits tamoxifen-resistant MCF-7 breast cancer cell growth through down-regulation of HER2 expression"

Article Title: Farnesoid X Receptor inhibits tamoxifen-resistant MCF-7 breast cancer cell growth through down-regulation of HER2 expression

Journal: Oncogene

doi: 10.1038/onc.2011.124

FXR expression and activation in MCF-7 and MCF-7TR1 cells. (a) Western blot analysis of HER2, EGFR, ERα in total protein extracts from MCF-7 and MCF-7TR1 cells; β-Actin was used as loading control. (b) Soft Agar growth assay in MCF-7 and MCF-7TR1 cells plated in 0.35% agarose and treated with EGF 100ng/ml in the presence or absence of Herceptin (10µg/ml). After 14 days of growth colonies > 50 µm diameter were counted. n.s. (nonsignificant); *p
Figure Legend Snippet: FXR expression and activation in MCF-7 and MCF-7TR1 cells. (a) Western blot analysis of HER2, EGFR, ERα in total protein extracts from MCF-7 and MCF-7TR1 cells; β-Actin was used as loading control. (b) Soft Agar growth assay in MCF-7 and MCF-7TR1 cells plated in 0.35% agarose and treated with EGF 100ng/ml in the presence or absence of Herceptin (10µg/ml). After 14 days of growth colonies > 50 µm diameter were counted. n.s. (nonsignificant); *p

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

12) Product Images from "Caffeine Promotes Ultraviolet B-induced Apoptosis in Human Keratinocytes without Complete DNA Repair *"

Article Title: Caffeine Promotes Ultraviolet B-induced Apoptosis in Human Keratinocytes without Complete DNA Repair *

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M111.222349

Caffeine inhibits UVB-induced AKT activation. A , immunoblot analysis of phospho-AKT (p-AKT), AKT, phospho-ERK (p-ERK), phospho-EGFR (p-EGFR), EGFR, and β-actin in cells pretreated with caffeine at indicated concentration at 1.5 h post-UVB (20
Figure Legend Snippet: Caffeine inhibits UVB-induced AKT activation. A , immunoblot analysis of phospho-AKT (p-AKT), AKT, phospho-ERK (p-ERK), phospho-EGFR (p-EGFR), EGFR, and β-actin in cells pretreated with caffeine at indicated concentration at 1.5 h post-UVB (20

Techniques Used: Activation Assay, Concentration Assay

13) Product Images from "Enhanced suppression of proliferation and migration in highly-metastatic lung cancer cells by combination of valproic acid and coumarin-3-carboxylic acid and its molecular mechanisms of action"

Article Title: Enhanced suppression of proliferation and migration in highly-metastatic lung cancer cells by combination of valproic acid and coumarin-3-carboxylic acid and its molecular mechanisms of action

Journal: Cytotechnology

doi: 10.1007/s10616-012-9513-7

Effects of combination of VPA and HCCA on receptor protein expressions and phosphorylation of pEGFR/EGFR (a) , pVEGFR2/VEGFR2 (b ), and p–c-Met/c-Met (c ) in LLC cells. The cells were treated with the indicated concentrations of VPA/V and HCCA/C
Figure Legend Snippet: Effects of combination of VPA and HCCA on receptor protein expressions and phosphorylation of pEGFR/EGFR (a) , pVEGFR2/VEGFR2 (b ), and p–c-Met/c-Met (c ) in LLC cells. The cells were treated with the indicated concentrations of VPA/V and HCCA/C

Techniques Used:

14) Product Images from "Lysophosphatidic acid receptor 2 and Gi/Src pathway mediate cell motility through cyclooxygenase 2 expression in CAOV-3 ovarian cancer cells"

Article Title: Lysophosphatidic acid receptor 2 and Gi/Src pathway mediate cell motility through cyclooxygenase 2 expression in CAOV-3 ovarian cancer cells

Journal: Experimental & Molecular Medicine

doi: 10.3858/emm.2008.40.6.607

Src is involved in EGFR transactivation. The cells were pretreated with pharmacological inhibitors of Src (PP2) or MMP (GM6001) for 1 h, followed by stimulation with 25 µM LPA. (A) The cell lysates were immunoblotted as described in materials
Figure Legend Snippet: Src is involved in EGFR transactivation. The cells were pretreated with pharmacological inhibitors of Src (PP2) or MMP (GM6001) for 1 h, followed by stimulation with 25 µM LPA. (A) The cell lysates were immunoblotted as described in materials

Techniques Used:

LPA induces COX-2 expression through Gi, EGFR and ERK. (A) The cells were serum starved overnight, and stimulated with LPA for indicated times. Cell lysates were analyzed by immunoblotted against anti-COX-1 or anti-COX-2 antibody. As a control, the lysate
Figure Legend Snippet: LPA induces COX-2 expression through Gi, EGFR and ERK. (A) The cells were serum starved overnight, and stimulated with LPA for indicated times. Cell lysates were analyzed by immunoblotted against anti-COX-1 or anti-COX-2 antibody. As a control, the lysate

Techniques Used: Expressing

LPA2 is responsible for phosphorylation of EGFR and ERK, as well as COX-2 expression. (A) Total RNA of CAOV-3 cells were reverse transcribed and amplified utilizing specific primers for each LPA receptors with LPA or not as described in Materials and
Figure Legend Snippet: LPA2 is responsible for phosphorylation of EGFR and ERK, as well as COX-2 expression. (A) Total RNA of CAOV-3 cells were reverse transcribed and amplified utilizing specific primers for each LPA receptors with LPA or not as described in Materials and

Techniques Used: Expressing, Amplification

15) Product Images from "Activation of AKT by hypoxia: a potential target for hypoxic tumors of the head and neck"

Article Title: Activation of AKT by hypoxia: a potential target for hypoxic tumors of the head and neck

Journal: BMC Cancer

doi: 10.1186/1471-2407-12-463

Correlation between in vitro and in vivo expression of EGFR/HER2 and pAKT/pSTAT3/pERK1/2 in 6 HNSCC lines. A ) Correlation between in vitro and in vivo expression of EGFR and HER2. B ) Correlation between in vitro and in vivo expression of pAKT, pERK1/2 and pSTAT3. Expression was assessed by western blot analysis and depicted in relative units. The integrated optical density (IOD) was measured and all values were normalized to those of α-tubulin by dividing the IOD value for that specific marker by the IOD value of α-tubulin. In vitro expression of UT-SCC5 was set as standard. Error bars represent standard error of the mean and all axes are in log scale. Correlations between in vitro and in vivo expression were assessed using the Spearman correlation test.
Figure Legend Snippet: Correlation between in vitro and in vivo expression of EGFR/HER2 and pAKT/pSTAT3/pERK1/2 in 6 HNSCC lines. A ) Correlation between in vitro and in vivo expression of EGFR and HER2. B ) Correlation between in vitro and in vivo expression of pAKT, pERK1/2 and pSTAT3. Expression was assessed by western blot analysis and depicted in relative units. The integrated optical density (IOD) was measured and all values were normalized to those of α-tubulin by dividing the IOD value for that specific marker by the IOD value of α-tubulin. In vitro expression of UT-SCC5 was set as standard. Error bars represent standard error of the mean and all axes are in log scale. Correlations between in vitro and in vivo expression were assessed using the Spearman correlation test.

Techniques Used: In Vitro, In Vivo, Expressing, Western Blot, Marker

In vitro and in vivo expression of EGFR, pEGFR, pAKT and AKT in 6 HNSCC lines. Cell lines were both cultured as cell lines ( in vitro ) and grown as xenograft tumors ( in vivo ) and expression levels were determined with western blot. Expression of α-tubulin was used as loading control. A ) In vitro expression of EGFR, pEGFR, pAKT and AKT. B ) In vivo expression of EGFR, pEGFR, pAKT and AKT. Number of harvested tumors ranged from 2 to 4 per cell line.
Figure Legend Snippet: In vitro and in vivo expression of EGFR, pEGFR, pAKT and AKT in 6 HNSCC lines. Cell lines were both cultured as cell lines ( in vitro ) and grown as xenograft tumors ( in vivo ) and expression levels were determined with western blot. Expression of α-tubulin was used as loading control. A ) In vitro expression of EGFR, pEGFR, pAKT and AKT. B ) In vivo expression of EGFR, pEGFR, pAKT and AKT. Number of harvested tumors ranged from 2 to 4 per cell line.

Techniques Used: In Vitro, In Vivo, Expressing, Cell Culture, Western Blot

16) Product Images from "IL-1beta Signals through the EGF Receptor and Activates Egr-1 through MMP-ADAM"

Article Title: IL-1beta Signals through the EGF Receptor and Activates Egr-1 through MMP-ADAM

Journal: PLoS ONE

doi: 10.1371/journal.pone.0039811

ADAM17 is required for IL-1beta–inducible Egr-1 expression. ( A ) Western blotting for Egr-1, ADAM17, EGFR, IL-1RI or beta-actin using total extracts of growth-quiescent wild-type or ADAM17-deficient mEFs treated with IL-1beta for 30 or 60 min. ( B ) Interaction of 125 I-IL-1beta with ADAM17WT and ADAM17-deficient cells. Growth-quiescent ADAM17WT and ADAM17−/− mEFs (1.8×10 4 cells/well) were incubated with increasing amounts of 125 I-IL-1beta in 1% BSA/PBS for 1 h at 4°C. The cells were washed and lysed with 1M NaOH prior to assessment of counts in an automated gamma-counter.
Figure Legend Snippet: ADAM17 is required for IL-1beta–inducible Egr-1 expression. ( A ) Western blotting for Egr-1, ADAM17, EGFR, IL-1RI or beta-actin using total extracts of growth-quiescent wild-type or ADAM17-deficient mEFs treated with IL-1beta for 30 or 60 min. ( B ) Interaction of 125 I-IL-1beta with ADAM17WT and ADAM17-deficient cells. Growth-quiescent ADAM17WT and ADAM17−/− mEFs (1.8×10 4 cells/well) were incubated with increasing amounts of 125 I-IL-1beta in 1% BSA/PBS for 1 h at 4°C. The cells were washed and lysed with 1M NaOH prior to assessment of counts in an automated gamma-counter.

Techniques Used: Expressing, Western Blot, Incubation

17) Product Images from "Cortactin Is a Substrate of Activated Cdc42-Associated Kinase 1 (ACK1) during Ligand-induced Epidermal Growth Factor Receptor Downregulation"

Article Title: Cortactin Is a Substrate of Activated Cdc42-Associated Kinase 1 (ACK1) during Ligand-induced Epidermal Growth Factor Receptor Downregulation

Journal: PLoS ONE

doi: 10.1371/journal.pone.0044363

EGFR downregulation requires ACK1 and the cortactin SH3 domain. (A) 1483 cells were transfected with non-targeting (Ctl) or human-specific ACK1 siRNA (ACK1 Si) for 48 h. Murine Myc-ACK1 was subsequently transfected into ACK depleted cells to rescue ACK1 expression. Cells were serum starved for 16 h and then treated with EGF for the indicated times. Following stimulation, clarified lysates were immunoprecipitated and immunoblotted with anti-EGFR antibodies. Total cell lysates were immunoblotted with anti-ACK1 and anti-actin antibodies. (B) 1483 cells were transfected with a non-targeting (Ctl) or cortactin specific siRNA (CTTN Si) for 48 h. Cortactin expression was rescued by transfection with FLAG-cortactin wild type (WT) or with an SH3-null binding mutant (W525K). Cells were serum starved for 16 h prior to EGF stimulation for the indicated times. EGFR was immunoprecipated and immunoblotted with anti-EGFR antibodies. Total cell lysates were immunoblotted with anti-cortactin to verify knockdown and expression of the FLAG-cortactin rescue constructs. Western blotting with anti-actin antibodies was conducted to verify equal protein loading. Blots are representative of two independent experiments.
Figure Legend Snippet: EGFR downregulation requires ACK1 and the cortactin SH3 domain. (A) 1483 cells were transfected with non-targeting (Ctl) or human-specific ACK1 siRNA (ACK1 Si) for 48 h. Murine Myc-ACK1 was subsequently transfected into ACK depleted cells to rescue ACK1 expression. Cells were serum starved for 16 h and then treated with EGF for the indicated times. Following stimulation, clarified lysates were immunoprecipitated and immunoblotted with anti-EGFR antibodies. Total cell lysates were immunoblotted with anti-ACK1 and anti-actin antibodies. (B) 1483 cells were transfected with a non-targeting (Ctl) or cortactin specific siRNA (CTTN Si) for 48 h. Cortactin expression was rescued by transfection with FLAG-cortactin wild type (WT) or with an SH3-null binding mutant (W525K). Cells were serum starved for 16 h prior to EGF stimulation for the indicated times. EGFR was immunoprecipated and immunoblotted with anti-EGFR antibodies. Total cell lysates were immunoblotted with anti-cortactin to verify knockdown and expression of the FLAG-cortactin rescue constructs. Western blotting with anti-actin antibodies was conducted to verify equal protein loading. Blots are representative of two independent experiments.

Techniques Used: Transfection, CTL Assay, Expressing, Immunoprecipitation, Binding Assay, Mutagenesis, Construct, Western Blot

Cortactin localizes with ACK1 in vesicles containing ligand-bound EGFR. (A) 1483 cells serum starved for 16 h were stimulated with 100 nanograms/milliter Alexa Fluor-488 conjugated EGF (green) for 30 min. Cells were fixed and labeled with phalloidin (Actin; pseudocolored white) and anti-EGFR antibodies (pseudocolored red). Cells were evaluated by confocal microscopy and images rotated 45° and 90° as indicated to demonstrate EGF/EGFR colocalization throughout the z-plane. (B) Serum starved (No Tx) 1483 cells were stimulated with FITC-EGF (pseudocolored white) as in (A). Cells were fixed and labeled with anti-ACK1 (green) and cortactin (red) antibodies. Confocal images of labeled EGR in the apical (top) and ventral (bottom) cellular regions are shown. Dashed boxes in the merged images indicate the areas enlarged in the photos to the right . Scale bars, 20 micrometers.
Figure Legend Snippet: Cortactin localizes with ACK1 in vesicles containing ligand-bound EGFR. (A) 1483 cells serum starved for 16 h were stimulated with 100 nanograms/milliter Alexa Fluor-488 conjugated EGF (green) for 30 min. Cells were fixed and labeled with phalloidin (Actin; pseudocolored white) and anti-EGFR antibodies (pseudocolored red). Cells were evaluated by confocal microscopy and images rotated 45° and 90° as indicated to demonstrate EGF/EGFR colocalization throughout the z-plane. (B) Serum starved (No Tx) 1483 cells were stimulated with FITC-EGF (pseudocolored white) as in (A). Cells were fixed and labeled with anti-ACK1 (green) and cortactin (red) antibodies. Confocal images of labeled EGR in the apical (top) and ventral (bottom) cellular regions are shown. Dashed boxes in the merged images indicate the areas enlarged in the photos to the right . Scale bars, 20 micrometers.

Techniques Used: Labeling, Confocal Microscopy

18) Product Images from "Non-Ligand-Induced Dimerization is Sufficient to Initiate the Signalling and Endocytosis of EGF Receptor"

Article Title: Non-Ligand-Induced Dimerization is Sufficient to Initiate the Signalling and Endocytosis of EGF Receptor

Journal: International Journal of Molecular Sciences

doi: 10.3390/ijms17081200

Stimulation of various signal transduction pathways by activation of EGFR-GFP or LZ-EGFR-GFP. ( A ) 293T cells were transiently transfected with EGFR-GFP or LZ-EGFR-GFP. Following serum starvation for 24 h, cells were treated with or without EGF. The cell lysates were subjected to immunoblotting analysis with rabbit anti-SHC, rabbit anti-phospho-PLC-γ1, rabbit anti-PLC-γ1, mouse anti-phospho-ERK1/2, mouse anti-Erk1/2, rabbit anti-phospho-Akt and rabbit anti-Akt antibodies; ( B ) Quantification of the data from ( A ). The band is quantitated by densitometry with image J software. The protein phosphorylation level of the control (EGFR-GFP, without EGF treatment) was set to 1 and the phosphorylation of the proteins under other conditions was expressed as the fold increase compared to control. Each value is the average of at least three independent experiments and the error bar is the standard error. **: p
Figure Legend Snippet: Stimulation of various signal transduction pathways by activation of EGFR-GFP or LZ-EGFR-GFP. ( A ) 293T cells were transiently transfected with EGFR-GFP or LZ-EGFR-GFP. Following serum starvation for 24 h, cells were treated with or without EGF. The cell lysates were subjected to immunoblotting analysis with rabbit anti-SHC, rabbit anti-phospho-PLC-γ1, rabbit anti-PLC-γ1, mouse anti-phospho-ERK1/2, mouse anti-Erk1/2, rabbit anti-phospho-Akt and rabbit anti-Akt antibodies; ( B ) Quantification of the data from ( A ). The band is quantitated by densitometry with image J software. The protein phosphorylation level of the control (EGFR-GFP, without EGF treatment) was set to 1 and the phosphorylation of the proteins under other conditions was expressed as the fold increase compared to control. Each value is the average of at least three independent experiments and the error bar is the standard error. **: p

Techniques Used: Transduction, Activation Assay, Transfection, Planar Chromatography, Software

19) Product Images from "Targeted Inhibition of Multiple Receptor Tyrosine Kinases in Mesothelioma 1Targeted Inhibition of Multiple Receptor Tyrosine Kinases in Mesothelioma 1 2"

Article Title: Targeted Inhibition of Multiple Receptor Tyrosine Kinases in Mesothelioma 1Targeted Inhibition of Multiple Receptor Tyrosine Kinases in Mesothelioma 1 2

Journal: Neoplasia (New York, N.Y.)

doi:

Effects of HSP90 inhibition (17-AAG) in mesothelioma cell lines by 17-AAG treatment for 48 hours in serum-containing medium. (A) EGFR, AXL, and MET expression. (B) p27, p53, and p21 expression and cleavage of apoptosis biomarker caspase 3. β-Actin is a loading control.
Figure Legend Snippet: Effects of HSP90 inhibition (17-AAG) in mesothelioma cell lines by 17-AAG treatment for 48 hours in serum-containing medium. (A) EGFR, AXL, and MET expression. (B) p27, p53, and p21 expression and cleavage of apoptosis biomarker caspase 3. β-Actin is a loading control.

Techniques Used: Inhibition, Expressing, Biomarker Assay

(A) Immunoblot analysis quantitations of EGFR, MET, and AXL expression in mesothelioma cells versus nonneoplastic mesothelial cells (HM3). The expression quantitations (lower panel) were normalized to the HM3 cells. (B) EGFR, MET, and AXL activation in mesothelioma cells was validated by immunoprecipitation of each kinase followed by phosphotyrosine immunoblot analysis.
Figure Legend Snippet: (A) Immunoblot analysis quantitations of EGFR, MET, and AXL expression in mesothelioma cells versus nonneoplastic mesothelial cells (HM3). The expression quantitations (lower panel) were normalized to the HM3 cells. (B) EGFR, MET, and AXL activation in mesothelioma cells was validated by immunoprecipitation of each kinase followed by phosphotyrosine immunoblot analysis.

Techniques Used: Expressing, Activation Assay, Immunoprecipitation

(A) Cell viability determined by the CellTiter-Glo ATP-based luminescence assay in mesothelioma cell lines after 72 hours of treatment with inhibitors of EGFR (1 µ M gefitinib), MET (1 µ M PHA-665752), AXL (1 µ M DP-3975), and HSP90 (1 µ M 17-AAG). Data were normalized to the DMSO control, and shown are mean values (±SD) of quadruplicate cultures. (B) Phospho-RTK arrays show concurrent inactivation of multiple RTKs in mesothelioma cell lines. After treatment with 17-AAG (1 µ M) for 6 hours in serum-free medium, phosphorylation quantitations (lower panel) were normalized to the negative controls.
Figure Legend Snippet: (A) Cell viability determined by the CellTiter-Glo ATP-based luminescence assay in mesothelioma cell lines after 72 hours of treatment with inhibitors of EGFR (1 µ M gefitinib), MET (1 µ M PHA-665752), AXL (1 µ M DP-3975), and HSP90 (1 µ M 17-AAG). Data were normalized to the DMSO control, and shown are mean values (±SD) of quadruplicate cultures. (B) Phospho-RTK arrays show concurrent inactivation of multiple RTKs in mesothelioma cell lines. After treatment with 17-AAG (1 µ M) for 6 hours in serum-free medium, phosphorylation quantitations (lower panel) were normalized to the negative controls.

Techniques Used: Luminescence Assay

Inactivation of multiple RTKs and signaling intermediates by HSP90 inhibition. (A) EGFR, AXL, and MET phosphorylation in cell immunoprecipitates after 6 hours of 17-AAG treatment in serum-free medium. (B) EGFR, ERBB3, MET, AKT, S6, and MAPK phosphorylation after 6 hours of 17-AAG treatment in serum-free medium. β-Actin stain is a loading control.
Figure Legend Snippet: Inactivation of multiple RTKs and signaling intermediates by HSP90 inhibition. (A) EGFR, AXL, and MET phosphorylation in cell immunoprecipitates after 6 hours of 17-AAG treatment in serum-free medium. (B) EGFR, ERBB3, MET, AKT, S6, and MAPK phosphorylation after 6 hours of 17-AAG treatment in serum-free medium. β-Actin stain is a loading control.

Techniques Used: Inhibition, Staining

Coactivation of multiple RTKs in mesothelioma cell lines. (A) Total cell lysates (500 µ g) from MESO924 cells grown for 2 hours in serum-containing (FBS+) or serum-free (FBS-) medium were analyzed by phospho-RTK array. Each RTK is spotted in duplicate, and the spots at each corner are positive controls, whereas eight spots at the lower right are negative controls. (B) Total cell lysates (500 µ g) from normal mesothelial cells grown for 2 hours in serum-free medium analyzed by phospho-RTK array. (C) Immunoblot evaluations of EGFR, ERBB3, MET, and AXL in MESO924 versus nonneoplastic mesothelial cells. Actin stain is a loading control.
Figure Legend Snippet: Coactivation of multiple RTKs in mesothelioma cell lines. (A) Total cell lysates (500 µ g) from MESO924 cells grown for 2 hours in serum-containing (FBS+) or serum-free (FBS-) medium were analyzed by phospho-RTK array. Each RTK is spotted in duplicate, and the spots at each corner are positive controls, whereas eight spots at the lower right are negative controls. (B) Total cell lysates (500 µ g) from normal mesothelial cells grown for 2 hours in serum-free medium analyzed by phospho-RTK array. (C) Immunoblot evaluations of EGFR, ERBB3, MET, and AXL in MESO924 versus nonneoplastic mesothelial cells. Actin stain is a loading control.

Techniques Used: Staining

20) Product Images from "Extracellular ATP stimulates epithelial cell motility through Pyk2-mediated activation of the EGF receptor"

Article Title: Extracellular ATP stimulates epithelial cell motility through Pyk2-mediated activation of the EGF receptor

Journal: Cellular signalling

doi: 10.1016/j.cellsig.2011.07.021

Knockdown of Pyk2 with siRNA inhibits EGFR activation after ATP stimulation but not after stimulation with EGF. (A) HCLE cells transfected with 10 nM of control or Pyk2 siRNA were treated with ATP as indicated. Immunoblots were probed for EGFR phosphorylated
Figure Legend Snippet: Knockdown of Pyk2 with siRNA inhibits EGFR activation after ATP stimulation but not after stimulation with EGF. (A) HCLE cells transfected with 10 nM of control or Pyk2 siRNA were treated with ATP as indicated. Immunoblots were probed for EGFR phosphorylated

Techniques Used: Activation Assay, Transfection, Western Blot

Expression of a Pyk2 dominant-negative mutant inhibits SFK and EGFR activation after wounding but not after stimulation with EGF. (A) Cells were infected with control adenovirus or adenovirus coding for PRNK and treated with ATP as indicated. Blots were
Figure Legend Snippet: Expression of a Pyk2 dominant-negative mutant inhibits SFK and EGFR activation after wounding but not after stimulation with EGF. (A) Cells were infected with control adenovirus or adenovirus coding for PRNK and treated with ATP as indicated. Blots were

Techniques Used: Expressing, Dominant Negative Mutation, Activation Assay, Infection

21) Product Images from "Distinct Activation of Epidermal Growth Factor Receptor by UTP Contributes to Epithelial Cell Wound Repair"

Article Title: Distinct Activation of Epidermal Growth Factor Receptor by UTP Contributes to Epithelial Cell Wound Repair

Journal: The American Journal of Pathology

doi: 10.1016/j.ajpath.2010.11.060

Recruitment and phosphorylation of EGFR differs in response to stimuli. A: The HCLEs were cultured to confluence and stimulated with control media change (−) over 60 minutes. The EGF (5 minutes) was used as a positive control (+). The pEGFR was detected using a phosphorylated tyrosine (p-tyr) antibody and is normalized to EGFR. B through D: The HCLEs were cultured to confluence and stimulated with EGF (0.5 and 5 nmol/L) or UTP (100 μmol/L) over 60 minutes. Confluent cells were lysed, and EGFR was immunoprecipitated from the lysates. The SDS-PAGE and Western blot analyses of the immunoprecipitation were performed for EGFR, Shc, Src, and Grb2; and probed for p-tyr to determine pEGFR. Images are representative of four experiments. E and F: Densitometric analysis [5.0-nmol/L EGF (black); 0.5-nmol/L EGF (light gray); and 100-μmol/L UTP (dark gray)]. Images are representative of four independent experiments.
Figure Legend Snippet: Recruitment and phosphorylation of EGFR differs in response to stimuli. A: The HCLEs were cultured to confluence and stimulated with control media change (−) over 60 minutes. The EGF (5 minutes) was used as a positive control (+). The pEGFR was detected using a phosphorylated tyrosine (p-tyr) antibody and is normalized to EGFR. B through D: The HCLEs were cultured to confluence and stimulated with EGF (0.5 and 5 nmol/L) or UTP (100 μmol/L) over 60 minutes. Confluent cells were lysed, and EGFR was immunoprecipitated from the lysates. The SDS-PAGE and Western blot analyses of the immunoprecipitation were performed for EGFR, Shc, Src, and Grb2; and probed for p-tyr to determine pEGFR. Images are representative of four experiments. E and F: Densitometric analysis [5.0-nmol/L EGF (black); 0.5-nmol/L EGF (light gray); and 100-μmol/L UTP (dark gray)]. Images are representative of four independent experiments.

Techniques Used: Cell Culture, Positive Control, Immunoprecipitation, SDS Page, Western Blot

The HCLEs were stimulated with HB-EGF. A: The HCLEs were cultured to confluence and stimulated with HB-EGF (1 nmol/L) over 60 minutes. The EGFR was immunoprecipitated from the lysates, and SDS-PAGE and Western blot analyses of the immunoprecipitation were performed. Antibodies directed against EGFR and Grb2 and an antibody to p-tyr were used. The pEGFR was normalized to EGFR. B: The HCLEs were cultured to confluence, preincubated for 20 minutes with a functional blocking antibody to HB-EGF (40 μg/ml) or control media (−), and stimulated with media change (−), EGF (1 nmol/L), HB-EGF (1 nmol/L), or UTP (100 μmol/L) for 5 minutes. Lysates were extracted, and EGFR was immunoprecipitated. The EGFR and p-tyr antibodies were used, and pEGFR was normalized to EGFR.
Figure Legend Snippet: The HCLEs were stimulated with HB-EGF. A: The HCLEs were cultured to confluence and stimulated with HB-EGF (1 nmol/L) over 60 minutes. The EGFR was immunoprecipitated from the lysates, and SDS-PAGE and Western blot analyses of the immunoprecipitation were performed. Antibodies directed against EGFR and Grb2 and an antibody to p-tyr were used. The pEGFR was normalized to EGFR. B: The HCLEs were cultured to confluence, preincubated for 20 minutes with a functional blocking antibody to HB-EGF (40 μg/ml) or control media (−), and stimulated with media change (−), EGF (1 nmol/L), HB-EGF (1 nmol/L), or UTP (100 μmol/L) for 5 minutes. Lysates were extracted, and EGFR was immunoprecipitated. The EGFR and p-tyr antibodies were used, and pEGFR was normalized to EGFR.

Techniques Used: Cell Culture, Immunoprecipitation, SDS Page, Western Blot, Functional Assay, Blocking Assay

Injury and UTP recruit specific docking proteins to the EGFR. Comparisons were made in two cell lines and primary epithelial cells. The HCLEs ( A ), primary corneal epithelial cells ( B ), and E1-PAEs ( C ) were stimulated with a control media change (C), injury (W), UTP (25 μmol/L), or EGF (5 nmol/L) for 5 minutes. Cells were lysed, and EGFR was immunoprecipitated from the lysates. SDS-PAGE and Western blot analyses of the immunoprecipitation were performed for EGFR, PLCγ1, Src, Shc, and Grb2. D: Phosphorylation of EGFR was determined after HCLEs were stimulated with media change (control), EGF (0.5 and 5 nmol/L), and UTP (100 μmol/L) at 5 and 60 minutes. SDS-PAGE and Western blot analyses of the immunoprecipitation were performed for EGFR. The pEGFR was detected using a phosphorylated tyrosine antibody. The pEGFR is normalized to EGFR. Data represent a minimum of three independent experiments.
Figure Legend Snippet: Injury and UTP recruit specific docking proteins to the EGFR. Comparisons were made in two cell lines and primary epithelial cells. The HCLEs ( A ), primary corneal epithelial cells ( B ), and E1-PAEs ( C ) were stimulated with a control media change (C), injury (W), UTP (25 μmol/L), or EGF (5 nmol/L) for 5 minutes. Cells were lysed, and EGFR was immunoprecipitated from the lysates. SDS-PAGE and Western blot analyses of the immunoprecipitation were performed for EGFR, PLCγ1, Src, Shc, and Grb2. D: Phosphorylation of EGFR was determined after HCLEs were stimulated with media change (control), EGF (0.5 and 5 nmol/L), and UTP (100 μmol/L) at 5 and 60 minutes. SDS-PAGE and Western blot analyses of the immunoprecipitation were performed for EGFR. The pEGFR was detected using a phosphorylated tyrosine antibody. The pEGFR is normalized to EGFR. Data represent a minimum of three independent experiments.

Techniques Used: Immunoprecipitation, SDS Page, Western Blot

22) Product Images from "Up-regulation of the kinase gene SGK1 by progesterone activates the AP-1–NDRG1 axis in both PR-positive and -negative breast cancer cells"

Article Title: Up-regulation of the kinase gene SGK1 by progesterone activates the AP-1–NDRG1 axis in both PR-positive and -negative breast cancer cells

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.RA118.002894

Depletion of NDRG1 activates multiple cellular kinases and increases migration of MDA-MB-231 cells. A , Western blot analysis depicting knockdown of NDRG1 in MD-231 breast cancer cells (PR-negative). sh-NT was used as vector control for NDRG1 expression. β-Actin protein was used as a loading control for Western blotting. Numbers on the blot indicate the intensity ratio for NDRG1, normalized to respective β-actin levels. The analysis is representative of three independent experiments. B , Western blot analysis of p-EGFR (Tyr-1086), p-AKT (Ser-473), and p-ERK1/2 (Thr-202/Tyr-204) in NDRG1 knockdown clones of MD231 cells. β-Actin was used as a loading control for Western blotting, and β-actin for the p-EGFR and p-ERK1/2 panels is the same. The numbers on the blot indicate average intensity ratio calculated from all of the three replicate experiments for phosphorylation levels of EGFR, AKT, and ERK1/2, normalized to respective total protein levels (EGFR, AKT, and ERK2). Western blot analysis is representative of three independent experiments. C , migration of cells was measured from 0 to 20 h by using a time-lapse wound healing assay. The bar plot represents percentage wound closure (average of the three biological replicate experiments), and the comparison was with respect to sh-NT. The figure is representative of three independent experiments performed in triplicates. p value was calculated using Student's unpaired t test. **, p
Figure Legend Snippet: Depletion of NDRG1 activates multiple cellular kinases and increases migration of MDA-MB-231 cells. A , Western blot analysis depicting knockdown of NDRG1 in MD-231 breast cancer cells (PR-negative). sh-NT was used as vector control for NDRG1 expression. β-Actin protein was used as a loading control for Western blotting. Numbers on the blot indicate the intensity ratio for NDRG1, normalized to respective β-actin levels. The analysis is representative of three independent experiments. B , Western blot analysis of p-EGFR (Tyr-1086), p-AKT (Ser-473), and p-ERK1/2 (Thr-202/Tyr-204) in NDRG1 knockdown clones of MD231 cells. β-Actin was used as a loading control for Western blotting, and β-actin for the p-EGFR and p-ERK1/2 panels is the same. The numbers on the blot indicate average intensity ratio calculated from all of the three replicate experiments for phosphorylation levels of EGFR, AKT, and ERK1/2, normalized to respective total protein levels (EGFR, AKT, and ERK2). Western blot analysis is representative of three independent experiments. C , migration of cells was measured from 0 to 20 h by using a time-lapse wound healing assay. The bar plot represents percentage wound closure (average of the three biological replicate experiments), and the comparison was with respect to sh-NT. The figure is representative of three independent experiments performed in triplicates. p value was calculated using Student's unpaired t test. **, p

Techniques Used: Migration, Multiple Displacement Amplification, Western Blot, Plasmid Preparation, Expressing, Clone Assay, Wound Healing Assay

NDRG1 regulates the activation of multiple cellular kinases and cell migration in T47D cells. A , Western blot analysis depicting knockdown of NDRG1 in T47D cells (PR-positive). sh-NT was used as vector control for NDRG1 expression. β-Actin protein was used as a loading control for Western blotting. Numbers on the blot indicate intensity ratio for NDRG1 expression normalized to respective β-actin levels. The analysis is representative of three independent experiments. B , Western blot analysis of p-EGFR (Tyr-1086), p-AKT (Ser-473), and p-ERK1/2 (Thr-202/Tyr-204) in NDRG1 knockdown clones of T47D cells. β-Actin used as a loading control for Western blotting. The figure is representative of three independent experiments. Numbers on the blot indicate average intensity ratio calculated from all of the three replicate experiments for phosphorylation levels of EGFR, AKT, and ERK1/2, normalized to the respective total protein levels (EGFR, AKT, and ERK2). Western blot analysis is representative of three independent experiments. C , cell migration analysis upon depletion of NDRG1 in T47D breast cancer cells. Cells were monitored by a time-lapse wound healing assay for 20 h. Cell migration from the initial to the 20-h time point was plotted as percentage wound closure (average of the three biological replicate experiments), and the comparison was with respect to sh-NT. The figure is representative of three independent experiments performed in triplicates. p value was calculated using Student's unpaired t test. ***, p
Figure Legend Snippet: NDRG1 regulates the activation of multiple cellular kinases and cell migration in T47D cells. A , Western blot analysis depicting knockdown of NDRG1 in T47D cells (PR-positive). sh-NT was used as vector control for NDRG1 expression. β-Actin protein was used as a loading control for Western blotting. Numbers on the blot indicate intensity ratio for NDRG1 expression normalized to respective β-actin levels. The analysis is representative of three independent experiments. B , Western blot analysis of p-EGFR (Tyr-1086), p-AKT (Ser-473), and p-ERK1/2 (Thr-202/Tyr-204) in NDRG1 knockdown clones of T47D cells. β-Actin used as a loading control for Western blotting. The figure is representative of three independent experiments. Numbers on the blot indicate average intensity ratio calculated from all of the three replicate experiments for phosphorylation levels of EGFR, AKT, and ERK1/2, normalized to the respective total protein levels (EGFR, AKT, and ERK2). Western blot analysis is representative of three independent experiments. C , cell migration analysis upon depletion of NDRG1 in T47D breast cancer cells. Cells were monitored by a time-lapse wound healing assay for 20 h. Cell migration from the initial to the 20-h time point was plotted as percentage wound closure (average of the three biological replicate experiments), and the comparison was with respect to sh-NT. The figure is representative of three independent experiments performed in triplicates. p value was calculated using Student's unpaired t test. ***, p

Techniques Used: Activation Assay, Migration, Western Blot, Plasmid Preparation, Expressing, Clone Assay, Wound Healing Assay

23) Product Images from "Endocytosis of ubiquitylation-deficient EGF receptor mutants via clathrin coated pits is mediated by ubiquitylation"

Article Title: Endocytosis of ubiquitylation-deficient EGF receptor mutants via clathrin coated pits is mediated by ubiquitylation

Journal: Traffic (Copenhagen, Denmark)

doi: 10.1111/tra.12314

TIR-FM imaging of EGFR endocytosis in MDA-MB-231 cells expressing endogenously labeled epsin1 and AP2 (A) Internalization of 125 I-EGF by parental MDA-MB-231 and MDA-MB-231/AP2-RFP/GFP-EPN1 cells. The cells were incubated with 1 ng/ml 125 I-EGF for the indicated times at 37°C, and the ratio of internalized and surface 125 I-EGF was determined and plotted against time. (B) Three-channel time-lapse TIR-FM imaging of cells before and after stimulation with EGF-A647 (20 ng/ml) was performed through 488 nm (GFP), 561 (RFP) and 640 (Alexa647) nm channels at 37°C. Single-channel and merged image at a 3-min time-point after EGF-A647 stimulation are shown. Insets represent high magnification merged images of the regions marked by white rectangles. Arrows show examples of co-localization of EGF-A647 with either Epsin1-GFP spots (white arrow), with both adaptor proteins (pink arrow) or AP2-RFP (yellow arrow). Scale bars, 10 μm. ( C ) Percent of spots that were labeled only with either AP2-RFP or GFP-epsin1 and co-localized with EGF-Rh of the total number of spots singularly labeled with AP2-RFP or GFP-epsin1 was measured in time-series of TIR-FM imaging performed as in ( A ). Image analysis of 805 tracks from 5 cells was performed using Imaris 7.1 software. Mean values for each time point with error bars representing standard deviation of the mean are plotted against time. ( D ) Representative examples of fluorescence traces and kymographs from time-series of AP2-RFP, GFP-epsin1 and EGF-A647 imaging in CCPs which hosted a scission event. TIR-FM imaging was performed as ( B ). A.u.f.i. , arbitrary units of fluorescence intensity.
Figure Legend Snippet: TIR-FM imaging of EGFR endocytosis in MDA-MB-231 cells expressing endogenously labeled epsin1 and AP2 (A) Internalization of 125 I-EGF by parental MDA-MB-231 and MDA-MB-231/AP2-RFP/GFP-EPN1 cells. The cells were incubated with 1 ng/ml 125 I-EGF for the indicated times at 37°C, and the ratio of internalized and surface 125 I-EGF was determined and plotted against time. (B) Three-channel time-lapse TIR-FM imaging of cells before and after stimulation with EGF-A647 (20 ng/ml) was performed through 488 nm (GFP), 561 (RFP) and 640 (Alexa647) nm channels at 37°C. Single-channel and merged image at a 3-min time-point after EGF-A647 stimulation are shown. Insets represent high magnification merged images of the regions marked by white rectangles. Arrows show examples of co-localization of EGF-A647 with either Epsin1-GFP spots (white arrow), with both adaptor proteins (pink arrow) or AP2-RFP (yellow arrow). Scale bars, 10 μm. ( C ) Percent of spots that were labeled only with either AP2-RFP or GFP-epsin1 and co-localized with EGF-Rh of the total number of spots singularly labeled with AP2-RFP or GFP-epsin1 was measured in time-series of TIR-FM imaging performed as in ( A ). Image analysis of 805 tracks from 5 cells was performed using Imaris 7.1 software. Mean values for each time point with error bars representing standard deviation of the mean are plotted against time. ( D ) Representative examples of fluorescence traces and kymographs from time-series of AP2-RFP, GFP-epsin1 and EGF-A647 imaging in CCPs which hosted a scission event. TIR-FM imaging was performed as ( B ). A.u.f.i. , arbitrary units of fluorescence intensity.

Techniques Used: Imaging, Multiple Displacement Amplification, Expressing, Labeling, Incubation, Software, Standard Deviation, Fluorescence

24) Product Images from "Nf2/Merlin controls progenitor homeostasis and tumorigenesis in the liver"

Article Title: Nf2/Merlin controls progenitor homeostasis and tumorigenesis in the liver

Journal: Genes & Development

doi: 10.1101/gad.1938710

Deregulated EGFR signaling drives the overproliferation of Nf2 −/− liver progenitors in vivo. ( A , B ) After only 10 d of erlotinib treatment, a reduction of the lesion area (outlined by dashed lies) is apparent in Alb-Cre;Nf2 lox/lox mice ( B ) compared with the Alb-Cre;Nf2 lox/lox littermates treated with vehicle 6% captisol ( A ). Hematoxylin and eosin-stained sections are shown (100×). Portal veins (arrowheads) and centrilobular veins (asterisks) are denoted. ( C , D ) Immunohistochemical detection of BrdU incorporation reveals a marked decrease in proliferating OCs within the lesions in erlotinib-treated Alb-Cre;Nf2 lox/lox mice ( D ) compared with the vehicle-treated littermates ( C ) (200×). A single BrdU pulse was given 2 h before sacrifice. ( E ) Quantification of the erlotinib-induced decrease in BrdU-incorporating OCs in vivo. For each liver sample, ∼3000 to ∼5000 OC nuclei were counted. The graph reflects the number of BrdU-positive nuclei per total nuclei within the lesions across multiple animals ( n = 4 for erlotinib group; n = 3 for vehicle group). (**) P
Figure Legend Snippet: Deregulated EGFR signaling drives the overproliferation of Nf2 −/− liver progenitors in vivo. ( A , B ) After only 10 d of erlotinib treatment, a reduction of the lesion area (outlined by dashed lies) is apparent in Alb-Cre;Nf2 lox/lox mice ( B ) compared with the Alb-Cre;Nf2 lox/lox littermates treated with vehicle 6% captisol ( A ). Hematoxylin and eosin-stained sections are shown (100×). Portal veins (arrowheads) and centrilobular veins (asterisks) are denoted. ( C , D ) Immunohistochemical detection of BrdU incorporation reveals a marked decrease in proliferating OCs within the lesions in erlotinib-treated Alb-Cre;Nf2 lox/lox mice ( D ) compared with the vehicle-treated littermates ( C ) (200×). A single BrdU pulse was given 2 h before sacrifice. ( E ) Quantification of the erlotinib-induced decrease in BrdU-incorporating OCs in vivo. For each liver sample, ∼3000 to ∼5000 OC nuclei were counted. The graph reflects the number of BrdU-positive nuclei per total nuclei within the lesions across multiple animals ( n = 4 for erlotinib group; n = 3 for vehicle group). (**) P

Techniques Used: In Vivo, Mouse Assay, Staining, Immunohistochemistry, BrdU Incorporation Assay

25) Product Images from "Role of Protein kinase C, Ca2+, Pyk2 and c-Src in Agonist Activation of Rat Lacrimal Gland p42/p44 MAPK."

Article Title: Role of Protein kinase C, Ca2+, Pyk2 and c-Src in Agonist Activation of Rat Lacrimal Gland p42/p44 MAPK.

Journal: FEBS letters

doi:

Effect of PMA on p42/p44 MAPK Activation Lacrimal gland acini were incubated with PMA (10 −6 M) for 0–30 min ( A ) or with PMA (10 −10 –10 −6 M) for 5 min ( B ) and the amount of activated p42/p44 MAPK/total p42 MAPK was measured. Insert blots are representative of 3–7 independent experiments. Data are mean ± SEM. * indicates statistical significance from t=0 or basal. ( C ) Acini were also incubated with EGF (10 −7 M) or PMA (10 −6 M) for 5 min, EGFR was immunoprecipitated and samples blotted for phosphotyrosine or EGFR.
Figure Legend Snippet: Effect of PMA on p42/p44 MAPK Activation Lacrimal gland acini were incubated with PMA (10 −6 M) for 0–30 min ( A ) or with PMA (10 −10 –10 −6 M) for 5 min ( B ) and the amount of activated p42/p44 MAPK/total p42 MAPK was measured. Insert blots are representative of 3–7 independent experiments. Data are mean ± SEM. * indicates statistical significance from t=0 or basal. ( C ) Acini were also incubated with EGF (10 −7 M) or PMA (10 −6 M) for 5 min, EGFR was immunoprecipitated and samples blotted for phosphotyrosine or EGFR.

Techniques Used: Activation Assay, Incubation, Immunoprecipitation

26) Product Images from "miR-3140 suppresses tumor cell growth by targeting BRD4 via its coding sequence and downregulates the BRD4-NUT fusion oncoprotein"

Article Title: miR-3140 suppresses tumor cell growth by targeting BRD4 via its coding sequence and downregulates the BRD4-NUT fusion oncoprotein

Journal: Scientific Reports

doi: 10.1038/s41598-018-22767-y

miR-3140 targeted CDK2 and EGFR by binding their 3′UTR regions. ( a ) Left, identification of downregulated genes after miR-3140 transfection by a gene expression array. The Venn diagram shows that 228 genes were commonly downregulated (fold change > 2) by transfection of miR-3140 in Panc1, MIAPaCa2, and MDA-MB-231 cells. Right, prediction of candidate target genes regulated by miR-3140 via their 3′UTR. The Venn diagram shows that 99 genes were predicted as candidate 3′UTR-targets of miR-3140 by the TargetScan program. ( b ) Western blot analysis of CDK2, CDK6, and EGFR in Panc1 and MIAPaCa2 cells 72 hours after transfection with 10 nmol/L of miR-NC or miR-3140 . ( c ) Luciferase reporter assays. Panc1 cells were transfected with the pmirGLO Dual Luciferase vectors containing wild type (Wt) CDK2 and EGFR or mutant (Mt) 3’UTR target sites of these genes, and after 6 hours, either miR-NC or miR-3140 was additionally transfected. Top, putative binding site of miR-3140 within the 3′UTR of each gene and mutant sequences. Bottom, results of the luciferase assay; * P
Figure Legend Snippet: miR-3140 targeted CDK2 and EGFR by binding their 3′UTR regions. ( a ) Left, identification of downregulated genes after miR-3140 transfection by a gene expression array. The Venn diagram shows that 228 genes were commonly downregulated (fold change > 2) by transfection of miR-3140 in Panc1, MIAPaCa2, and MDA-MB-231 cells. Right, prediction of candidate target genes regulated by miR-3140 via their 3′UTR. The Venn diagram shows that 99 genes were predicted as candidate 3′UTR-targets of miR-3140 by the TargetScan program. ( b ) Western blot analysis of CDK2, CDK6, and EGFR in Panc1 and MIAPaCa2 cells 72 hours after transfection with 10 nmol/L of miR-NC or miR-3140 . ( c ) Luciferase reporter assays. Panc1 cells were transfected with the pmirGLO Dual Luciferase vectors containing wild type (Wt) CDK2 and EGFR or mutant (Mt) 3’UTR target sites of these genes, and after 6 hours, either miR-NC or miR-3140 was additionally transfected. Top, putative binding site of miR-3140 within the 3′UTR of each gene and mutant sequences. Bottom, results of the luciferase assay; * P

Techniques Used: Binding Assay, Transfection, Expressing, Multiple Displacement Amplification, Western Blot, Luciferase, Mutagenesis

27) Product Images from "Use of monoclonal antibody-IRDye800CW bioconjugates in the resection of breast cancer"

Article Title: Use of monoclonal antibody-IRDye800CW bioconjugates in the resection of breast cancer

Journal: The Journal of surgical research

doi: 10.1016/j.jss.2013.11.1089

(A) Western blot of 2LMP receptor expression. EGFR was the most strongly expressed within the 2LMP cell line. VEGF showed moderate expression, HER2/neu had low-level expression, and IL-6R expression was not demonstrated. (B–F) Antigen binding
Figure Legend Snippet: (A) Western blot of 2LMP receptor expression. EGFR was the most strongly expressed within the 2LMP cell line. VEGF showed moderate expression, HER2/neu had low-level expression, and IL-6R expression was not demonstrated. (B–F) Antigen binding

Techniques Used: Western Blot, Expressing, Binding Assay

28) Product Images from "Single Vesicle Analysis of Endocytic Fission on Microtubules In Vitro"

Article Title: Single Vesicle Analysis of Endocytic Fission on Microtubules In Vitro

Journal: Traffic (Copenhagen, Denmark)

doi: 10.1111/j.1600-0854.2008.00725.x

Segregation of Transferrin receptor from ntcp, ASOR from ASGPR and ASOR from EGFR during microtubule-based motility in vitro
Figure Legend Snippet: Segregation of Transferrin receptor from ntcp, ASOR from ASGPR and ASOR from EGFR during microtubule-based motility in vitro

Techniques Used: In Vitro

29) Product Images from "TIEG1 Inhibits Breast Cancer Invasion and Metastasis by Inhibition of Epidermal Growth Factor Receptor (EGFR) Transcription and the EGFR Signaling Pathway"

Article Title: TIEG1 Inhibits Breast Cancer Invasion and Metastasis by Inhibition of Epidermal Growth Factor Receptor (EGFR) Transcription and the EGFR Signaling Pathway

Journal: Molecular and Cellular Biology

doi: 10.1128/MCB.06152-11

Overexpression of TIEG1 attenuates EGFR expression and histone acetylation of the EGFR promoter in MDA-MB-231HM cells. (A and B) TIEG1 expression vector or control vector was transfected into MDA-MB-231HM cells and generated stable transfectants, MDA-MB-231HM/TIEG1
Figure Legend Snippet: Overexpression of TIEG1 attenuates EGFR expression and histone acetylation of the EGFR promoter in MDA-MB-231HM cells. (A and B) TIEG1 expression vector or control vector was transfected into MDA-MB-231HM cells and generated stable transfectants, MDA-MB-231HM/TIEG1

Techniques Used: Over Expression, Expressing, Multiple Displacement Amplification, Plasmid Preparation, Transfection, Generated

TIEG1 inhibits EGFR promoter activity in MDA-MB-231HM and MDA-MB-468 cells. (A) Model depicting binding of TIEG1 transcriptional factor to the Sp1 sites on the EGFR promoter. (B and C) TIEG1 and HDAC1 inhibited EGFR promoter activity in MDA-MB-231HM and
Figure Legend Snippet: TIEG1 inhibits EGFR promoter activity in MDA-MB-231HM and MDA-MB-468 cells. (A) Model depicting binding of TIEG1 transcriptional factor to the Sp1 sites on the EGFR promoter. (B and C) TIEG1 and HDAC1 inhibited EGFR promoter activity in MDA-MB-231HM and

Techniques Used: Activity Assay, Multiple Displacement Amplification, Binding Assay

TIEG1 expression is associated with EGFR expression and metastasis in breast tumors.
Figure Legend Snippet: TIEG1 expression is associated with EGFR expression and metastasis in breast tumors.

Techniques Used: Expressing

Binding status of TIEG1 complex and histone acetylation on the EGFR promoter in MDA-MB-231 and MDA-MB-231HM cells. (A and B) Relative TIEG1 and EGFR mRNA levels were detected in MDA-MB-231 and MDA-MB-231HM cells. Total RNA was extracted from the cells
Figure Legend Snippet: Binding status of TIEG1 complex and histone acetylation on the EGFR promoter in MDA-MB-231 and MDA-MB-231HM cells. (A and B) Relative TIEG1 and EGFR mRNA levels were detected in MDA-MB-231 and MDA-MB-231HM cells. Total RNA was extracted from the cells

Techniques Used: Binding Assay, Multiple Displacement Amplification

TIEG1 is associated with EGFR expression in breast cancer tissues. (A) TIEG1 and EGFR expression in breast cancer tissues. Immunohistochemical staining was carried out on histological sections in 90 pairs of breast cancer tissues with the anti-TIEG1 or
Figure Legend Snippet: TIEG1 is associated with EGFR expression in breast cancer tissues. (A) TIEG1 and EGFR expression in breast cancer tissues. Immunohistochemical staining was carried out on histological sections in 90 pairs of breast cancer tissues with the anti-TIEG1 or

Techniques Used: Expressing, Immunohistochemistry, Staining

TIEG1 inhibits breast cancer cell invasion by inhibition of EGFR signaling pathway. (A) Overexpression of TIEG1 inhibited the EGFR signaling pathway. TIEG1 expression vector or control vector was transfected into MDA-MB-231HM or MDA-MB-468 cells and generated
Figure Legend Snippet: TIEG1 inhibits breast cancer cell invasion by inhibition of EGFR signaling pathway. (A) Overexpression of TIEG1 inhibited the EGFR signaling pathway. TIEG1 expression vector or control vector was transfected into MDA-MB-231HM or MDA-MB-468 cells and generated

Techniques Used: Inhibition, Over Expression, Expressing, Plasmid Preparation, Transfection, Multiple Displacement Amplification, Generated

TIEG1 inhibits breast cancer metastasis by inhibition of EGFR signaling pathway. (A and B) MDA-MB-231HM/TIEG1, MDA-MB-231HM/Vector, MDA-MB-231, MDA-MB-468/TIEG1, and MDA-MB-468/Vector cells, as indicated, were injected into the mammary fat pad of athymic
Figure Legend Snippet: TIEG1 inhibits breast cancer metastasis by inhibition of EGFR signaling pathway. (A and B) MDA-MB-231HM/TIEG1, MDA-MB-231HM/Vector, MDA-MB-231, MDA-MB-468/TIEG1, and MDA-MB-468/Vector cells, as indicated, were injected into the mammary fat pad of athymic

Techniques Used: Inhibition, Multiple Displacement Amplification, Plasmid Preparation, Injection

Knockdown of TIEG1 induces EGFR expression in MDA-MB-231 cells. (A and B) Knockdown of TIEG1 in MDA-MB-231 cells induced EGFR expression. MDA-MB-231 cells were treated with 100 nM TIEG1 siRNA or nontargeting siRNA for 48 h. Real-time PCR (A) and Western
Figure Legend Snippet: Knockdown of TIEG1 induces EGFR expression in MDA-MB-231 cells. (A and B) Knockdown of TIEG1 in MDA-MB-231 cells induced EGFR expression. MDA-MB-231 cells were treated with 100 nM TIEG1 siRNA or nontargeting siRNA for 48 h. Real-time PCR (A) and Western

Techniques Used: Expressing, Multiple Displacement Amplification, Real-time Polymerase Chain Reaction, Western Blot

TIEG1 expression is associated with EGFR expression and metastasis in breast tumors.
Figure Legend Snippet: TIEG1 expression is associated with EGFR expression and metastasis in breast tumors.

Techniques Used: Expressing

30) Product Images from "Loss of functional MYO1C/myosin 1c, a motor protein involved in lipid raft trafficking, disrupts autophagosome-lysosome fusion"

Article Title: Loss of functional MYO1C/myosin 1c, a motor protein involved in lipid raft trafficking, disrupts autophagosome-lysosome fusion

Journal: Autophagy

doi: 10.4161/15548627.2014.984272

( See previous page ). Analysis of lysosome function in MYO1C-depleted cells. ( A ) Mock, MYO1C-depleted cells, and bafilomycin A 1 -treated cells were incubated with the LysoTracker Red probe and imaged using live cell microscopy to monitor lysosomal pH. Swollen lysosomes in MYO1C knockdown cell appear to be acidic. ( B ) To determine lysosomal enzyme activity in live cells control and MYO1C-depleted HeLa cells were incubated with Magic Red. Confocal microscopy was used to image cathepsin–associated hydrolysis of Magic Red into its red fluorescent form. ( C ) Calculation of cathepsin activity using Volocity Imaging software revealed no significant difference between cathepsin activity in control and MYO1C-knockdown cells. Values are means ± s.e.m from 3 independent experiments ( > 1900 cells). ns, not significant. ( D ) To measure EGFR degradation in control and MYO1C siRNA-treated HeLa cells, cells were serum-starved overnight, and incubated with 100 μg/ml cycloheximide for 2 h before stimulating with 100 ng/ml EGF for 0, 1, 2 and 3 h. EGFR levels were quantified from immunoblots and normalized to a loading control. No significant difference in the rate of EGFR degradation was observed in control and knockdown cells. Values are means ± s.e.m from 3 independent knockdown experiments.
Figure Legend Snippet: ( See previous page ). Analysis of lysosome function in MYO1C-depleted cells. ( A ) Mock, MYO1C-depleted cells, and bafilomycin A 1 -treated cells were incubated with the LysoTracker Red probe and imaged using live cell microscopy to monitor lysosomal pH. Swollen lysosomes in MYO1C knockdown cell appear to be acidic. ( B ) To determine lysosomal enzyme activity in live cells control and MYO1C-depleted HeLa cells were incubated with Magic Red. Confocal microscopy was used to image cathepsin–associated hydrolysis of Magic Red into its red fluorescent form. ( C ) Calculation of cathepsin activity using Volocity Imaging software revealed no significant difference between cathepsin activity in control and MYO1C-knockdown cells. Values are means ± s.e.m from 3 independent experiments ( > 1900 cells). ns, not significant. ( D ) To measure EGFR degradation in control and MYO1C siRNA-treated HeLa cells, cells were serum-starved overnight, and incubated with 100 μg/ml cycloheximide for 2 h before stimulating with 100 ng/ml EGF for 0, 1, 2 and 3 h. EGFR levels were quantified from immunoblots and normalized to a loading control. No significant difference in the rate of EGFR degradation was observed in control and knockdown cells. Values are means ± s.e.m from 3 independent knockdown experiments.

Techniques Used: Polyacrylamide Gel Electrophoresis, Incubation, Microscopy, Activity Assay, Confocal Microscopy, Imaging, Software, Western Blot

31) Product Images from "EGFRvIII/integrin β3 interaction in hypoxic and vitronectinenriching microenvironment promote GBM progression and metastasis"

Article Title: EGFRvIII/integrin β3 interaction in hypoxic and vitronectinenriching microenvironment promote GBM progression and metastasis

Journal: Oncotarget

doi: 10.18632/oncotarget.6730

Significance of EGFRvIII, integrin β3, HIF-1α, VTN, and their relations in malignant glioma samples ( A ) Kaplan-Meier analyses [38] on the correlations between the relative expression of ITGB3 , HIF-1α , or VTN (vitronectin), and the overall survival time of 97 patients with malignant glioma at the mRNA level. ( B ) Pearson correlation on the transcriptional level of ITGB3 , HIF-1α , or VTN , and EGFRvIII in these tumor samples was analyzed by R software. Significance of EGFRvIII , ITGB3 and EGFR in these tumor samples were analyzed by bioinformatics methods as indicated in Materials and Methods. In the gene sets regulated by EGFRvIII (labeled in red) ( C ), or ITGB3 ( D ), or EGFR ( E ); effector expressions of the integrin β3/EGFRvIII-associated signaling axis in Figure 4E were shown; the effectors that had a significant change in gene expression were labeled with *.
Figure Legend Snippet: Significance of EGFRvIII, integrin β3, HIF-1α, VTN, and their relations in malignant glioma samples ( A ) Kaplan-Meier analyses [38] on the correlations between the relative expression of ITGB3 , HIF-1α , or VTN (vitronectin), and the overall survival time of 97 patients with malignant glioma at the mRNA level. ( B ) Pearson correlation on the transcriptional level of ITGB3 , HIF-1α , or VTN , and EGFRvIII in these tumor samples was analyzed by R software. Significance of EGFRvIII , ITGB3 and EGFR in these tumor samples were analyzed by bioinformatics methods as indicated in Materials and Methods. In the gene sets regulated by EGFRvIII (labeled in red) ( C ), or ITGB3 ( D ), or EGFR ( E ); effector expressions of the integrin β3/EGFRvIII-associated signaling axis in Figure 4E were shown; the effectors that had a significant change in gene expression were labeled with *.

Techniques Used: Expressing, Software, Labeling

32) Product Images from "Adenovirus vector-based incorporation of a photo-cross-linkable amino acid into proteins in human primary cells and cancerous cell lines"

Article Title: Adenovirus vector-based incorporation of a photo-cross-linkable amino acid into proteins in human primary cells and cancerous cell lines

Journal: Scientific Reports

doi: 10.1038/srep36946

Photo-cross-linking between GRB2(mTmdZLys109) and endogenous cell-signalling proteins in human MDA-MB-468 cells. ( a ) A structural model of mTmdZLys (magenta sticks) in place of Lys109 of GRB2 (ribbons) bound to a tyrosine-phosphorylated peptide derived from EGFR (green sticks); the model was constructed by modifying the crystal structure data obtained from Protein Data Bank (code 1ZFP). ( b – d ) Western-blot analyses of cross-linked products. GRB2 proteins, tagged with FLAG peptide, were precipitated from protein fractions of the human cells treated as indicated. Then, western blotting was performed using anti-GRB2 antibody ( b ), anti-EGFR antibody ( c ), and anti-SHC antibody ( d ). Black arrows indicate GBR2 ( b ), EGFR ( c ), and SHC ( d ). Red and blue arrows indicate probable cross-linked complexes of GRB2 with EGFR and SHC, respectively.
Figure Legend Snippet: Photo-cross-linking between GRB2(mTmdZLys109) and endogenous cell-signalling proteins in human MDA-MB-468 cells. ( a ) A structural model of mTmdZLys (magenta sticks) in place of Lys109 of GRB2 (ribbons) bound to a tyrosine-phosphorylated peptide derived from EGFR (green sticks); the model was constructed by modifying the crystal structure data obtained from Protein Data Bank (code 1ZFP). ( b – d ) Western-blot analyses of cross-linked products. GRB2 proteins, tagged with FLAG peptide, were precipitated from protein fractions of the human cells treated as indicated. Then, western blotting was performed using anti-GRB2 antibody ( b ), anti-EGFR antibody ( c ), and anti-SHC antibody ( d ). Black arrows indicate GBR2 ( b ), EGFR ( c ), and SHC ( d ). Red and blue arrows indicate probable cross-linked complexes of GRB2 with EGFR and SHC, respectively.

Techniques Used: Multiple Displacement Amplification, Derivative Assay, Construct, Western Blot

Photo-cross-linking between GRB2(mTmdZLys109) and endogenous cell-signalling proteins in human HUVEC cells. The GRB2 proteins, tagged with FLAG peptide, were precipitated from protein fractions of the human cells treated as indicated. Then, western blotting was performed using anti-EGFR antibody ( a ) and anti-SHC antibody ( b ). Black arrows indicate EGFR ( a ) and SHC ( b ). Red and blue arrows indicate probable cross-linked complexes of GRB2 with EGFR and SHC, respectively. The dashed arrow indicates non-specific binders for GRB2.
Figure Legend Snippet: Photo-cross-linking between GRB2(mTmdZLys109) and endogenous cell-signalling proteins in human HUVEC cells. The GRB2 proteins, tagged with FLAG peptide, were precipitated from protein fractions of the human cells treated as indicated. Then, western blotting was performed using anti-EGFR antibody ( a ) and anti-SHC antibody ( b ). Black arrows indicate EGFR ( a ) and SHC ( b ). Red and blue arrows indicate probable cross-linked complexes of GRB2 with EGFR and SHC, respectively. The dashed arrow indicates non-specific binders for GRB2.

Techniques Used: Western Blot

33) Product Images from "A Small Insulinomimetic Molecule Also Improves Insulin Sensitivity in Diabetic Mice"

Article Title: A Small Insulinomimetic Molecule Also Improves Insulin Sensitivity in Diabetic Mice

Journal: PLoS ONE

doi: 10.1371/journal.pone.0169809

dmp binding to IR augments insulin signalling pathway. (A) dmp fails to activate EGFR. L6 myotubes or 3T3L1 adipocytes were treated with or without 250 nM dmp for 4h. The cell lysates were analyzed by immunoblotting with anti-pEGFR and anti-EGFR antibodies. (B,C) dmp can induce IR phosphorylation in a dose dependent manner. L6 myotubes were treated with insulin (20–120 n m ) or dmp (50–300 n m ) for 4h and IR phosphorylation was monitored by ELISA (B) or immunoblotting with anti-pIR and anti-IR antibodies (C). (D) IR kinase activity was determined in L6 myotubes which were incubated with varied concentrations of insulin or dmp. (E) dmp stimulates IR and its downstream kinases phosphorylation. L6 myotubes or 3T3L1 adipocytes were treated with or without Insulin (100 nM) or dmp (250 nM) for 4h and the IR phosphorylation and its downstream signalling were monitored by immunoblotting. (F) L6 myotubes transfected with GFP-GLUT4 chimeric gene were incubated with insulin (100 nM) or dmp (250nM) for 4h. Cells on the cover slips were fixed in paraformaldehyde and observed under florescent microscope for GFP-GLUT4 translocation. (G) dmp like insulin promotes glucose uptake. L6 myotubes or skeletal muscle cells from soleus muscle of neonatal mice (2-3days) were incubated with 100 n m insulin or 250 n m dmp for 25 min. [ 14 C] 2-DOG was then added, and the cells were further incubated for 5 min. [ 14 C] 2-DOG uptake was measured by scintillation counting. * P
Figure Legend Snippet: dmp binding to IR augments insulin signalling pathway. (A) dmp fails to activate EGFR. L6 myotubes or 3T3L1 adipocytes were treated with or without 250 nM dmp for 4h. The cell lysates were analyzed by immunoblotting with anti-pEGFR and anti-EGFR antibodies. (B,C) dmp can induce IR phosphorylation in a dose dependent manner. L6 myotubes were treated with insulin (20–120 n m ) or dmp (50–300 n m ) for 4h and IR phosphorylation was monitored by ELISA (B) or immunoblotting with anti-pIR and anti-IR antibodies (C). (D) IR kinase activity was determined in L6 myotubes which were incubated with varied concentrations of insulin or dmp. (E) dmp stimulates IR and its downstream kinases phosphorylation. L6 myotubes or 3T3L1 adipocytes were treated with or without Insulin (100 nM) or dmp (250 nM) for 4h and the IR phosphorylation and its downstream signalling were monitored by immunoblotting. (F) L6 myotubes transfected with GFP-GLUT4 chimeric gene were incubated with insulin (100 nM) or dmp (250nM) for 4h. Cells on the cover slips were fixed in paraformaldehyde and observed under florescent microscope for GFP-GLUT4 translocation. (G) dmp like insulin promotes glucose uptake. L6 myotubes or skeletal muscle cells from soleus muscle of neonatal mice (2-3days) were incubated with 100 n m insulin or 250 n m dmp for 25 min. [ 14 C] 2-DOG was then added, and the cells were further incubated for 5 min. [ 14 C] 2-DOG uptake was measured by scintillation counting. * P

Techniques Used: Binding Assay, Enzyme-linked Immunosorbent Assay, Activity Assay, Incubation, Transfection, Microscopy, Translocation Assay, Mouse Assay

34) Product Images from "Integrin ?5 contributes to the tumorigenic potential of breast cancer cells through Src-FAK and MEK-ERK signaling pathways"

Article Title: Integrin ?5 contributes to the tumorigenic potential of breast cancer cells through Src-FAK and MEK-ERK signaling pathways

Journal: Oncogene

doi: 10.1038/onc.2012.320

Integrin β5 mediates activation FAK and ERK signaling (A) Immunoblot analysis of whole-cell protein extracts from control, shB5 and shB5-over-expressing integrin β5 MDA-MB-231 cells treated with TGF-β1 (2ng/ml) for 24h (upper panel) or 2h (lower panel). Membranes were probed with antibodies for FAK, phospho-FAK-Tyr861, phospho-paxillin-Ty31, paxillin, ERK1/2, phospho-ERK1/2, phospho-Smad2, and GAPDH as loading control. (B) Immunoblot analysis of whole-cell protein extracts from control and siRNA-ITGB5-treated BT549, MCF7, and T47D cells. Membranes were probed with antibodies for integrin β5, phospho-FAK-Tyr861, FAK, ERK 1/2, phospho-ERK1/2 and a-tubulin. (C) MDA-MB-231 (upper panel) and MCF10A (lower panel) cells were treated with the EGFR inhibitor AG1478 for 2h at the indicated concentrations. Immunoblot analysis was performed on whole-cell extracts probing for FAK, phospho-FAK-Tyr397, phospho-FAK-Tyr861, ERK1/2, and phospho-ERK1/2. (D) Immunoblot analysis of whole-cell protein extracts from control and shB5 cells, treated with EGF (100ng/ml) for 2h. Where indicated, a 1h pre-treatment with AG1478 (5μM) was performed. Membranes were probed for phospho - EGFR-Tyr845, total EGFR, phospho - ERK1/2, and total ERK1/2. (E) Immunoblotting of whole-cell protein extracts from control cells, treated with 100ng/ml EGF for 2h. Where indicated, a 1h pre-treatment with 100nM Dasatinib was done. Membranes were probed for phospho - EGFR-Tyr845 and EGFR.
Figure Legend Snippet: Integrin β5 mediates activation FAK and ERK signaling (A) Immunoblot analysis of whole-cell protein extracts from control, shB5 and shB5-over-expressing integrin β5 MDA-MB-231 cells treated with TGF-β1 (2ng/ml) for 24h (upper panel) or 2h (lower panel). Membranes were probed with antibodies for FAK, phospho-FAK-Tyr861, phospho-paxillin-Ty31, paxillin, ERK1/2, phospho-ERK1/2, phospho-Smad2, and GAPDH as loading control. (B) Immunoblot analysis of whole-cell protein extracts from control and siRNA-ITGB5-treated BT549, MCF7, and T47D cells. Membranes were probed with antibodies for integrin β5, phospho-FAK-Tyr861, FAK, ERK 1/2, phospho-ERK1/2 and a-tubulin. (C) MDA-MB-231 (upper panel) and MCF10A (lower panel) cells were treated with the EGFR inhibitor AG1478 for 2h at the indicated concentrations. Immunoblot analysis was performed on whole-cell extracts probing for FAK, phospho-FAK-Tyr397, phospho-FAK-Tyr861, ERK1/2, and phospho-ERK1/2. (D) Immunoblot analysis of whole-cell protein extracts from control and shB5 cells, treated with EGF (100ng/ml) for 2h. Where indicated, a 1h pre-treatment with AG1478 (5μM) was performed. Membranes were probed for phospho - EGFR-Tyr845, total EGFR, phospho - ERK1/2, and total ERK1/2. (E) Immunoblotting of whole-cell protein extracts from control cells, treated with 100ng/ml EGF for 2h. Where indicated, a 1h pre-treatment with 100nM Dasatinib was done. Membranes were probed for phospho - EGFR-Tyr845 and EGFR.

Techniques Used: Activation Assay, Expressing, Multiple Displacement Amplification

35) Product Images from "Mutant p53 initiates a feedback loop that involves Egr-1/EGF receptor/ERK in prostate cancer cells"

Article Title: Mutant p53 initiates a feedback loop that involves Egr-1/EGF receptor/ERK in prostate cancer cells

Journal: Oncogene

doi: 10.1038/onc.2010.24

Secretion of cytokines in prostate cancer cells (Panel A) DU145 cells were maintained in serum-free medium for three days. The conditioned medium was collected and added to 22Rv1 cells that had been maintained in serum-free medium for 24 hrs. After the indicated times, 22Rv1 cells were lysed and protein expression and phosphorylation was analyzed by western blot. P-Akt: anti-phospho-Akt. (Panel B) The experiment was performed as in panel A except that EGFR inhibitor PD168393 (1μM) was added 2 hours prior to the conditioned medium. (Panel C) Cells were plated at a controlled density and cultured in serum-free medium for three days. The absolute quantity of various cytokines from the conditioned medium of each cell line was measured by multiplex protein array. The western blot shown as insert compares Egr-1, p53 and phospho-ERK1/2 levels in these four cells lines. (Panel D) Cells were treated with siRNA-Egr1 for 48 hrs before RNA purification. Levels of mRNA were measured by quantitative real-time RT-PCR.
Figure Legend Snippet: Secretion of cytokines in prostate cancer cells (Panel A) DU145 cells were maintained in serum-free medium for three days. The conditioned medium was collected and added to 22Rv1 cells that had been maintained in serum-free medium for 24 hrs. After the indicated times, 22Rv1 cells were lysed and protein expression and phosphorylation was analyzed by western blot. P-Akt: anti-phospho-Akt. (Panel B) The experiment was performed as in panel A except that EGFR inhibitor PD168393 (1μM) was added 2 hours prior to the conditioned medium. (Panel C) Cells were plated at a controlled density and cultured in serum-free medium for three days. The absolute quantity of various cytokines from the conditioned medium of each cell line was measured by multiplex protein array. The western blot shown as insert compares Egr-1, p53 and phospho-ERK1/2 levels in these four cells lines. (Panel D) Cells were treated with siRNA-Egr1 for 48 hrs before RNA purification. Levels of mRNA were measured by quantitative real-time RT-PCR.

Techniques Used: Expressing, Western Blot, Cell Culture, Multiplex Assay, Protein Array, Purification, Quantitative RT-PCR

p53 regulates the expression of EGFR (Panel A, left) DU145 cells were treated with Pifithrin-α at the indicated concentrations for 16 hrs before cell lysis. Results were analyzed by western blot using the indicated antibodies. (Panel A, right) Cells were treated with pifithrin-α (50 μM) for 24 hours. RNA was purified and analyzed by RT-PCR followed by agarose gel electrophoresis. (Panel B) ChIP assay: DU145 were grown under normal conditions before being fixed with para-formaldehyde and submitted to ChIP. Non-specific IgG (NS) were used as a negative control. Antibodies against Polymerase II, p53 and Egr-1 were used to capture the protein-DNA complexes. Genomic DNA was used as input. The EGFR promoter was amplified by PCR and analyzed by agarose electrophoresis. (Panel C) Cells were transfected with expression plasmid pCMV-Egr1 or with the transfection reagent alone (Mock). RNA was purified 72 hrs later and analyzed by RT-PCR. Duplicates are shown. (Panel D) The experiment was performed as described in figure 6A . Controls showing p53 and actin mRNA are the same as in figure 6A .
Figure Legend Snippet: p53 regulates the expression of EGFR (Panel A, left) DU145 cells were treated with Pifithrin-α at the indicated concentrations for 16 hrs before cell lysis. Results were analyzed by western blot using the indicated antibodies. (Panel A, right) Cells were treated with pifithrin-α (50 μM) for 24 hours. RNA was purified and analyzed by RT-PCR followed by agarose gel electrophoresis. (Panel B) ChIP assay: DU145 were grown under normal conditions before being fixed with para-formaldehyde and submitted to ChIP. Non-specific IgG (NS) were used as a negative control. Antibodies against Polymerase II, p53 and Egr-1 were used to capture the protein-DNA complexes. Genomic DNA was used as input. The EGFR promoter was amplified by PCR and analyzed by agarose electrophoresis. (Panel C) Cells were transfected with expression plasmid pCMV-Egr1 or with the transfection reagent alone (Mock). RNA was purified 72 hrs later and analyzed by RT-PCR. Duplicates are shown. (Panel D) The experiment was performed as described in figure 6A . Controls showing p53 and actin mRNA are the same as in figure 6A .

Techniques Used: Expressing, Lysis, Western Blot, Purification, Reverse Transcription Polymerase Chain Reaction, Agarose Gel Electrophoresis, Chromatin Immunoprecipitation, Negative Control, Amplification, Polymerase Chain Reaction, Electrophoresis, Transfection, Plasmid Preparation

Constitutive activation of EGFR and ERK1/2 regulates Egr-1 expression (Panel A) DU145 and 22Rv1 cells were treated with PD168393 (2 μM) for 2 hrs. Cells were lysed and subjected to immunoprecipitation using anti-EGFR antibodies. Buffer alone (NS) or non-specific immunoglobulins (IgG) were negative controls. Whole cell lysates were loaded for positive control (input). (Panel B) DU145 (left) were treated with EGFR inhibitor PD168393 at 2 μM for the indicated times. 22Rv1 (right) were treated with PD168393 at 2 μM for 24 hrs. (Panel C) DU145 were incubated with increasing amounts of neutralizing anti-EGFR antibodies for 24 hours before lysis. (Panel D) Cells were treated with MEK inhibitor PD98059 for 2 hrs before lysis. All experiments were analyzed by western blot using the indicated antibodies. Actin or pan-ERK1 antibodies were used for loading controls. P-ERK: anti-phospho-ERK1/2; P-Tyr: anti-phospho-tyrosine.
Figure Legend Snippet: Constitutive activation of EGFR and ERK1/2 regulates Egr-1 expression (Panel A) DU145 and 22Rv1 cells were treated with PD168393 (2 μM) for 2 hrs. Cells were lysed and subjected to immunoprecipitation using anti-EGFR antibodies. Buffer alone (NS) or non-specific immunoglobulins (IgG) were negative controls. Whole cell lysates were loaded for positive control (input). (Panel B) DU145 (left) were treated with EGFR inhibitor PD168393 at 2 μM for the indicated times. 22Rv1 (right) were treated with PD168393 at 2 μM for 24 hrs. (Panel C) DU145 were incubated with increasing amounts of neutralizing anti-EGFR antibodies for 24 hours before lysis. (Panel D) Cells were treated with MEK inhibitor PD98059 for 2 hrs before lysis. All experiments were analyzed by western blot using the indicated antibodies. Actin or pan-ERK1 antibodies were used for loading controls. P-ERK: anti-phospho-ERK1/2; P-Tyr: anti-phospho-tyrosine.

Techniques Used: Activation Assay, Expressing, Immunoprecipitation, Positive Control, Incubation, Lysis, Western Blot

Egr-1 transcription induced by mutant p53 is mediated by MEK/ERK signaling (panel A) 22Rv1cells were mock-transfected or transfected with p53-V143A or p53-R175H. 72 hours later, cells were incubated with PD98059 for 4 hours. Total RNA was purified and mRNA levels were analyzed by RT-PCR. (panel B) 22Rv1cells were mock-transfected or transfected with wt-p53, p53-V143A or p53-R175H. 72 hours later, cells were lysed and analyzed by western blot. (panel C) The experiment was performed as in panel A except that EGFR inhibitor was added for 24 hours before RNA purification.
Figure Legend Snippet: Egr-1 transcription induced by mutant p53 is mediated by MEK/ERK signaling (panel A) 22Rv1cells were mock-transfected or transfected with p53-V143A or p53-R175H. 72 hours later, cells were incubated with PD98059 for 4 hours. Total RNA was purified and mRNA levels were analyzed by RT-PCR. (panel B) 22Rv1cells were mock-transfected or transfected with wt-p53, p53-V143A or p53-R175H. 72 hours later, cells were lysed and analyzed by western blot. (panel C) The experiment was performed as in panel A except that EGFR inhibitor was added for 24 hours before RNA purification.

Techniques Used: Mutagenesis, Transfection, Incubation, Purification, Reverse Transcription Polymerase Chain Reaction, Western Blot

36) Product Images from "Targeting the insulin-like growth factor receptor and Src signaling network for the treatment of non-small cell lung cancer"

Article Title: Targeting the insulin-like growth factor receptor and Src signaling network for the treatment of non-small cell lung cancer

Journal: Molecular Cancer

doi: 10.1186/s12943-015-0392-3

Transactivation of IGF-1R by activated Src. ( a ) H226B and H226Br cells were transiently transfected with empty or pcDNA3.1-Src (Y527F) vectors. ( b ) A549, H460, and H522 cells were serum-starved and then stimulated with EGF (50 ng/ml). ( c ) H520 cells were transfected with empty or pBabe-Puro EGFR WT vectors, treated with dasatinib (Dasa; 0.5 μM) for 2 h, and then stimulated with EGF (50 ng/ml) for 2 min. ( d ) A549 cells were transfected with scrambled (siCon) or Src siRNA (siSrc) and stimulated with EGF (50 ng/ml) for 5 min. ( e ) H226B cells were transfected with empty or pIRES2-EGFP-integrin β3 vectors, treated with dasatinib (Dasa; 0.5 μM) for 2 h, and then attached to fibronectin (FN)-coated dishes for 30 min. ( f , g ) In vitro Src kinase assay was performed using Src, either from recombinant protein (rSrc) or from immunoprecipitates (IP) from A549 cells untransfected ( f ) or from H226B cells transfected with wild-type or kinase-dead mutant Src (Y416F) ( g ), and recombinant IGF-1R (GST-IGF-1R) as a substrate. ( h ) H520 cells were transfected with empty, wild-type, or mutant IGF-1R (Y1135F)-expressing vectors. ( i ) A549 cells were serum-starved and then stimulated with IGF (100 ng/ml) for 5 minutes. ( j ) H1299 cells stably transfected with control- or IGF-1R shRNAs were stimulated with 10 % FBS for 5 minutes. ( k ) In vitro IGF-1R kinase assay was performed using IGF-1R immunoprecipitates (IP) from A549 cells and recombinant GST-Src as a substrate. The expression levels of the indicated proteins were determined by Western blot analysis
Figure Legend Snippet: Transactivation of IGF-1R by activated Src. ( a ) H226B and H226Br cells were transiently transfected with empty or pcDNA3.1-Src (Y527F) vectors. ( b ) A549, H460, and H522 cells were serum-starved and then stimulated with EGF (50 ng/ml). ( c ) H520 cells were transfected with empty or pBabe-Puro EGFR WT vectors, treated with dasatinib (Dasa; 0.5 μM) for 2 h, and then stimulated with EGF (50 ng/ml) for 2 min. ( d ) A549 cells were transfected with scrambled (siCon) or Src siRNA (siSrc) and stimulated with EGF (50 ng/ml) for 5 min. ( e ) H226B cells were transfected with empty or pIRES2-EGFP-integrin β3 vectors, treated with dasatinib (Dasa; 0.5 μM) for 2 h, and then attached to fibronectin (FN)-coated dishes for 30 min. ( f , g ) In vitro Src kinase assay was performed using Src, either from recombinant protein (rSrc) or from immunoprecipitates (IP) from A549 cells untransfected ( f ) or from H226B cells transfected with wild-type or kinase-dead mutant Src (Y416F) ( g ), and recombinant IGF-1R (GST-IGF-1R) as a substrate. ( h ) H520 cells were transfected with empty, wild-type, or mutant IGF-1R (Y1135F)-expressing vectors. ( i ) A549 cells were serum-starved and then stimulated with IGF (100 ng/ml) for 5 minutes. ( j ) H1299 cells stably transfected with control- or IGF-1R shRNAs were stimulated with 10 % FBS for 5 minutes. ( k ) In vitro IGF-1R kinase assay was performed using IGF-1R immunoprecipitates (IP) from A549 cells and recombinant GST-Src as a substrate. The expression levels of the indicated proteins were determined by Western blot analysis

Techniques Used: Transfection, In Vitro, Kinase Assay, Recombinant, Mutagenesis, Expressing, Stable Transfection, Western Blot

37) Product Images from "Notch1 serves as a prognostic factor and regulates metastasis via regulating EGFR expression in hypopharyngeal squamous cell carcinoma"

Article Title: Notch1 serves as a prognostic factor and regulates metastasis via regulating EGFR expression in hypopharyngeal squamous cell carcinoma

Journal: OncoTargets and therapy

doi: 10.2147/OTT.S175423

The expression of EGFR and YBX1 were significantly inhibited after si-Notch1 transfection. Notes: ( A and B ) The RNA levels of EGFR and YBX1 by real-time PCR assay (* P
Figure Legend Snippet: The expression of EGFR and YBX1 were significantly inhibited after si-Notch1 transfection. Notes: ( A and B ) The RNA levels of EGFR and YBX1 by real-time PCR assay (* P

Techniques Used: Expressing, Transfection, Real-time Polymerase Chain Reaction

Ectopic expression of YBX1 resulted in substantial reversion on the effectiveness of Notch1 knockdown. Notes: ( A ) Further evidence showed that YBX1 upregulation antagonized Notch1 knockdown-induced inhibition on EGFR. The Western blot assay was used to record EGFR, YBX1, and p-YBX1 expression in FaDu cells co-transfected with si-Notch1 and YBX1 genes (* P
Figure Legend Snippet: Ectopic expression of YBX1 resulted in substantial reversion on the effectiveness of Notch1 knockdown. Notes: ( A ) Further evidence showed that YBX1 upregulation antagonized Notch1 knockdown-induced inhibition on EGFR. The Western blot assay was used to record EGFR, YBX1, and p-YBX1 expression in FaDu cells co-transfected with si-Notch1 and YBX1 genes (* P

Techniques Used: Expressing, Inhibition, Western Blot, Transfection

38) Product Images from "miR-217 and CAGE form feedback loop and regulates the response to anti-cancer drugs through EGFR and HER2"

Article Title: miR-217 and CAGE form feedback loop and regulates the response to anti-cancer drugs through EGFR and HER2

Journal: Oncotarget

doi: 10.18632/oncotarget.7185

HER2 is necessary for the interaction between CAGE and EGFR, and regulates the response to anti-cancer drugs ( A ) Malme3M R cells were transiently transfected with the indicated siRNA (each at 10 nM). At 24 h after transfection, cells were then treated with various concentrations of taxol, gefitinib or trastuzumab for 24 h, followed by MTT assays. ( B ) Malme3M R cells were transiently transfected with the indicated siRNA (10 nM). At 24 h after transfection, cells were then treated with various concentrations of taxol (1 μM), gefitinib (1 μM) or trastuzumab (10 μg/ml) for 24 h, followed by caspase-3 activity assays. ** p
Figure Legend Snippet: HER2 is necessary for the interaction between CAGE and EGFR, and regulates the response to anti-cancer drugs ( A ) Malme3M R cells were transiently transfected with the indicated siRNA (each at 10 nM). At 24 h after transfection, cells were then treated with various concentrations of taxol, gefitinib or trastuzumab for 24 h, followed by MTT assays. ( B ) Malme3M R cells were transiently transfected with the indicated siRNA (10 nM). At 24 h after transfection, cells were then treated with various concentrations of taxol (1 μM), gefitinib (1 μM) or trastuzumab (10 μg/ml) for 24 h, followed by caspase-3 activity assays. ** p

Techniques Used: Transfection, MTT Assay, Activity Assay

CAGE interacts with EGFR and is necessary for the increased phosphorylation of EGFR in Malme3M R cells ( A ) Malme3M cells were treated with taxol (1 μM) for various time intervals or cells were treated with various concentrations of taxol for 24 h. Cell lysates were subjected to immunoblot analysis. ( B ) Malme3M (1 × 10 6 ) or Malme3M R cells (1 × 10 6 ) were injected into the dorsal flank area of athymic nude mouse. Taxol (1 mg/kg) was injected into each nude mouse after the tumor reached a certain size (∼50 mm 3 ). Tumor volume was measured on the same day as injection of taxol. Tumor-bearing mice were assessed for weight loss. Five mice were used for the injection of each cell line. Each value represents an average obtained from five mice of each group. Data are expressed as mean ± SD. Immunoblot of tumor lysates was performed. Immunohistochemistry staining (right panel) of tumor tissue derived from Malme3M or Malme3M R cells was performed as described. Paraffin sections (4–6 μm thickness) of the tumor tissues were stained with the indicated antibodies. Immunohistochemistry staining employing secondary antibody alone served as a negative control. Representative images from five animals from each experimental group are shown (magnification, 400X; Olympus). H E staining was performed to check structural integrity. All animal experiments were approved by Institutional review Board for animal studies of Kangwon National University. ( C ) Malme3M R cells were treated with IgG (10 μg/ml), cetuximab (10 μg/ml) or gefitinib (1 μM) for 24 h. Cell lysates prepared were subjected to immunoblot and immunoprecipitation analysis. Cell lysates isolated from Malme3M R-As-CAGE cells that stably express anti-sense CAGE were also subjected to immunoblot and immunoprecipitation analysis. ( D ) Malme3M R cells were treated with IgG (10 μg/ml) or cetuximab (10 μg/ml) for 24 h. Cell lysates were immunoprecipitated with the indicated antibody (2 μg/ml), followed by immunoblot analysis (lower panel). Cell lysates were also subjected to immunoblot analysis (upper panel). ( E ) Immunofluorescence staining employing the indicated antibody (2 μg/ml) was performed as described.
Figure Legend Snippet: CAGE interacts with EGFR and is necessary for the increased phosphorylation of EGFR in Malme3M R cells ( A ) Malme3M cells were treated with taxol (1 μM) for various time intervals or cells were treated with various concentrations of taxol for 24 h. Cell lysates were subjected to immunoblot analysis. ( B ) Malme3M (1 × 10 6 ) or Malme3M R cells (1 × 10 6 ) were injected into the dorsal flank area of athymic nude mouse. Taxol (1 mg/kg) was injected into each nude mouse after the tumor reached a certain size (∼50 mm 3 ). Tumor volume was measured on the same day as injection of taxol. Tumor-bearing mice were assessed for weight loss. Five mice were used for the injection of each cell line. Each value represents an average obtained from five mice of each group. Data are expressed as mean ± SD. Immunoblot of tumor lysates was performed. Immunohistochemistry staining (right panel) of tumor tissue derived from Malme3M or Malme3M R cells was performed as described. Paraffin sections (4–6 μm thickness) of the tumor tissues were stained with the indicated antibodies. Immunohistochemistry staining employing secondary antibody alone served as a negative control. Representative images from five animals from each experimental group are shown (magnification, 400X; Olympus). H E staining was performed to check structural integrity. All animal experiments were approved by Institutional review Board for animal studies of Kangwon National University. ( C ) Malme3M R cells were treated with IgG (10 μg/ml), cetuximab (10 μg/ml) or gefitinib (1 μM) for 24 h. Cell lysates prepared were subjected to immunoblot and immunoprecipitation analysis. Cell lysates isolated from Malme3M R-As-CAGE cells that stably express anti-sense CAGE were also subjected to immunoblot and immunoprecipitation analysis. ( D ) Malme3M R cells were treated with IgG (10 μg/ml) or cetuximab (10 μg/ml) for 24 h. Cell lysates were immunoprecipitated with the indicated antibody (2 μg/ml), followed by immunoblot analysis (lower panel). Cell lysates were also subjected to immunoblot analysis (upper panel). ( E ) Immunofluorescence staining employing the indicated antibody (2 μg/ml) was performed as described.

Techniques Used: Injection, Mouse Assay, Immunohistochemistry, Staining, Derivative Assay, Negative Control, Immunoprecipitation, Isolation, Stable Transfection, Immunofluorescence

The inactivation of EGFR confers sensitivity to anti-cancer drugs and inhibits interactions of EGFR with CAGE and HER2 ( A ) Malme3M R cells were treated with the indicated peptide (each at 10 μM) for 48 h. Cell lysates were then isolated and subjected to immunoprecipitation and immunoblot analysis (left panel). Malme3M cells were transfected with the indicated construct (each at 1 μg) along with the indicated peptide (10 μM). At 48 h after transfection, cell lysates were subjected to immunoblot analysis (right panel). ( B ) Malme3M R cells were treated with the indicated peptide (each at 10 μM) for 48 h. Cells were then treated with various concentrations of trastuzumab or gefitnib for 24 h, followed by MTT assays. * p
Figure Legend Snippet: The inactivation of EGFR confers sensitivity to anti-cancer drugs and inhibits interactions of EGFR with CAGE and HER2 ( A ) Malme3M R cells were treated with the indicated peptide (each at 10 μM) for 48 h. Cell lysates were then isolated and subjected to immunoprecipitation and immunoblot analysis (left panel). Malme3M cells were transfected with the indicated construct (each at 1 μg) along with the indicated peptide (10 μM). At 48 h after transfection, cell lysates were subjected to immunoblot analysis (right panel). ( B ) Malme3M R cells were treated with the indicated peptide (each at 10 μM) for 48 h. Cells were then treated with various concentrations of trastuzumab or gefitnib for 24 h, followed by MTT assays. * p

Techniques Used: Isolation, Immunoprecipitation, Transfection, Construct, MTT Assay

CAGE confers resistance to EGFR inhibitors ( A ) Shows CAGE deletion constructs. ( B ) Each construct (1 μg) was transiently transfected into Malme3M cells. At 48 h after transfection, cell lysates were immunoprecipitated with the indicated antibody (2 μg/ml), followed by immunoblot analysis. Cell lysates were also subjected to immunoblot analysis, ( C ) Malme3M R cells were transiently transfected with the indicated siRNA (each at 10 nM). At 48 h after transfection, cell lysates were subjected to immunoblot analysis. ( D ) Each construct (1 μg) was transiently transfected into Malme3M cells. At 24 h after transfection, cells were then treated with the indicated anti-cancer drugs for 24 h, followed by MTT assays.
Figure Legend Snippet: CAGE confers resistance to EGFR inhibitors ( A ) Shows CAGE deletion constructs. ( B ) Each construct (1 μg) was transiently transfected into Malme3M cells. At 48 h after transfection, cell lysates were immunoprecipitated with the indicated antibody (2 μg/ml), followed by immunoblot analysis. Cell lysates were also subjected to immunoblot analysis, ( C ) Malme3M R cells were transiently transfected with the indicated siRNA (each at 10 nM). At 48 h after transfection, cell lysates were subjected to immunoblot analysis. ( D ) Each construct (1 μg) was transiently transfected into Malme3M cells. At 24 h after transfection, cells were then treated with the indicated anti-cancer drugs for 24 h, followed by MTT assays.

Techniques Used: Construct, Transfection, Immunoprecipitation, MTT Assay

39) Product Images from "Anti-claudin-4 extracellular domain antibody enhances the antitumoral effects of chemotherapeutic and antibody drugs in colorectal cancer"

Article Title: Anti-claudin-4 extracellular domain antibody enhances the antitumoral effects of chemotherapeutic and antibody drugs in colorectal cancer

Journal: Oncotarget

doi: 10.18632/oncotarget.26427

Antitumoral effect of 4D3 anti-CLDN4 antibody in human CRC cells ( A , C ) 4D3 enhances the growth inhibitory effects of 5-FU in HT29 cells (A) and Caco-2 cells (C). ( B , D ) 4D3 increases the effective intracellular concentration of 5-FU in HT29 cells (B) and Caco-2 cells (D). ( E , F ) Effect of 4D3 on EGFR phosphorylation (E) and HIF-1α expression (F) in HT29 cells. ( G , H ) Effect of treatment with 5-FU and/or 4D3 without TNFα (G) or with TNFα pretreatment (10 ng/mL, for 2 days) (H). n = 5 per group. ( I ) Effect of TNFα on growth of HT29 cells. Graph legend; TNFα (ng/mL). ( J ) Effect of TNFα on the growth inhibition induced by concurrent treatment with 5-FU and 4D3. Error bars, SD.
Figure Legend Snippet: Antitumoral effect of 4D3 anti-CLDN4 antibody in human CRC cells ( A , C ) 4D3 enhances the growth inhibitory effects of 5-FU in HT29 cells (A) and Caco-2 cells (C). ( B , D ) 4D3 increases the effective intracellular concentration of 5-FU in HT29 cells (B) and Caco-2 cells (D). ( E , F ) Effect of 4D3 on EGFR phosphorylation (E) and HIF-1α expression (F) in HT29 cells. ( G , H ) Effect of treatment with 5-FU and/or 4D3 without TNFα (G) or with TNFα pretreatment (10 ng/mL, for 2 days) (H). n = 5 per group. ( I ) Effect of TNFα on growth of HT29 cells. Graph legend; TNFα (ng/mL). ( J ) Effect of TNFα on the growth inhibition induced by concurrent treatment with 5-FU and 4D3. Error bars, SD.

Techniques Used: Concentration Assay, Expressing, Inhibition

40) Product Images from "β-Arrestin-1 Drives Endothelin-1–Mediated Podocyte Activation and Sustains Renal Injury"

Article Title: β-Arrestin-1 Drives Endothelin-1–Mediated Podocyte Activation and Sustains Renal Injury

Journal: Journal of the American Society of Nephrology : JASN

doi: 10.1681/ASN.2013040362

ET A R and β -arrestin-1 form a molecular signaling complex with Src promoting EGFR transactivation in ET-1-treated podocytes. (A) The association of β -arrestin-1 ( β -arr1) with ET A R and Src was studied in cell lysates from control and ET-1–-treated podocytes by immunoprecipitation (IP) assays using irrelevant IgG (negative control) or an anti– β -arr1 antibody followed by immunoblotting (IB) with anti-ET A R, anti-Src, and anti– β -arr1 antibodies. (B) The role of ET A R in the formation of the molecular signaling complex was assessed by IPs using irrelevant IgG or an anti–arr1 antibody in lysates from podocytes exposed to control medium, ET-1 (100 nM, 15 minutes) alone or in the presence of BQ123 (1 μ M), followed by IB with anti– β -arr1 and anti-Src antibodies. (C) Cell lysates from podocytes incubated with control medium or ET-1 (100 nM) for 5, 15, or 30 minutes were immunoblotted with anti–phospho-Src (Y416), anti-Src, anti–phospho-EGFR (Y845), anti-EGFR, anti–phospho-AKT, anti-AKT, anti–phospho-p42/44 MAPK, anti-p42/44 MAPK, and anti-HSP70 antibodies. (D) IB analysis using anti–phospho-EGFR (Y845) and anti-EGFR antibodies in podocytes exposed to control medium or stimulated with ET-1 (100 nM, 15 minutes) in the presence or absence of PP1 (1 μ M). EGFR phosphorylation status was also assessed in ET-1–treated podocytes silenced for β -arrestin-1 (si- β -arr1) by specific siRNA or transfected with scrambled siRNA (SCR). For all panels, data are representative of at least three experiments. Molecular mass is indicated in kilodaltons.
Figure Legend Snippet: ET A R and β -arrestin-1 form a molecular signaling complex with Src promoting EGFR transactivation in ET-1-treated podocytes. (A) The association of β -arrestin-1 ( β -arr1) with ET A R and Src was studied in cell lysates from control and ET-1–-treated podocytes by immunoprecipitation (IP) assays using irrelevant IgG (negative control) or an anti– β -arr1 antibody followed by immunoblotting (IB) with anti-ET A R, anti-Src, and anti– β -arr1 antibodies. (B) The role of ET A R in the formation of the molecular signaling complex was assessed by IPs using irrelevant IgG or an anti–arr1 antibody in lysates from podocytes exposed to control medium, ET-1 (100 nM, 15 minutes) alone or in the presence of BQ123 (1 μ M), followed by IB with anti– β -arr1 and anti-Src antibodies. (C) Cell lysates from podocytes incubated with control medium or ET-1 (100 nM) for 5, 15, or 30 minutes were immunoblotted with anti–phospho-Src (Y416), anti-Src, anti–phospho-EGFR (Y845), anti-EGFR, anti–phospho-AKT, anti-AKT, anti–phospho-p42/44 MAPK, anti-p42/44 MAPK, and anti-HSP70 antibodies. (D) IB analysis using anti–phospho-EGFR (Y845) and anti-EGFR antibodies in podocytes exposed to control medium or stimulated with ET-1 (100 nM, 15 minutes) in the presence or absence of PP1 (1 μ M). EGFR phosphorylation status was also assessed in ET-1–treated podocytes silenced for β -arrestin-1 (si- β -arr1) by specific siRNA or transfected with scrambled siRNA (SCR). For all panels, data are representative of at least three experiments. Molecular mass is indicated in kilodaltons.

Techniques Used: Immunoprecipitation, Negative Control, Incubation, Transfection

The complex ET A R/ β -arrestin-1/Src and the EGFR transactivation are required for β-catenin stability. (A) Cell lysates from control or ET-1–treated podocytes were immunoprecipitated with irrelevant IgG (negative control) or anti– β -arrestin-1 ( β -arr1) antibody and analyzed by immunoblotting (IB) using anti– β -catenin ( β -cat) and anti– β -arr1 antibodies. (B) Active β -cat was assessed in podocytes exposed to control medium, ET-1 (100 nM) alone or with BQ123 (1 μ M) by IB. Anti-HSP70 antibody was used to confirm equal protein loading. (C) Active β -cat in podocytes transfected with scrambled (SCR) or β -arrestin-1 (si- β -arr1) siRNA before exposure to ET-1 (100 nM) for 15 minutes. Anti-HSP70 antibody was used to confirm equal protein loading. (D) Lysates of podocytes treated with control medium, ET-1 (100 nM, 15 minutes) alone or in the presence of PP1 (1 μ M) were immunoprecipitated with irrelevant IgG or anti– β -cat antibody and analyzed by IB using an antiphosphorylated tyrosine (pTYR) and anti– β -cat antibodies. (E) Lysates of podocytes treated with control medium, ET-1 (100 nM, 15 minutes) alone or in the presence of AG1478 (1 μ M) were immunoprecipitated with irrelevant IgG or anti– β -cat antibody and analyzed by IB using anti-pTYR and anti– β -cat antibodies. (F) Accumulation of active β -cat was evaluated in podocytes exposed to control medium, ET-1 (100 nM) alone or supplemented with AG1478 (1 μ M). Cell lysates were analyzed by IB using anti-active β -cat and anti-HSP70 antibodies. For all panels, data are representative of at least two experiments. Molecular mass is indicated in kilodaltons.
Figure Legend Snippet: The complex ET A R/ β -arrestin-1/Src and the EGFR transactivation are required for β-catenin stability. (A) Cell lysates from control or ET-1–treated podocytes were immunoprecipitated with irrelevant IgG (negative control) or anti– β -arrestin-1 ( β -arr1) antibody and analyzed by immunoblotting (IB) using anti– β -catenin ( β -cat) and anti– β -arr1 antibodies. (B) Active β -cat was assessed in podocytes exposed to control medium, ET-1 (100 nM) alone or with BQ123 (1 μ M) by IB. Anti-HSP70 antibody was used to confirm equal protein loading. (C) Active β -cat in podocytes transfected with scrambled (SCR) or β -arrestin-1 (si- β -arr1) siRNA before exposure to ET-1 (100 nM) for 15 minutes. Anti-HSP70 antibody was used to confirm equal protein loading. (D) Lysates of podocytes treated with control medium, ET-1 (100 nM, 15 minutes) alone or in the presence of PP1 (1 μ M) were immunoprecipitated with irrelevant IgG or anti– β -cat antibody and analyzed by IB using an antiphosphorylated tyrosine (pTYR) and anti– β -cat antibodies. (E) Lysates of podocytes treated with control medium, ET-1 (100 nM, 15 minutes) alone or in the presence of AG1478 (1 μ M) were immunoprecipitated with irrelevant IgG or anti– β -cat antibody and analyzed by IB using anti-pTYR and anti– β -cat antibodies. (F) Accumulation of active β -cat was evaluated in podocytes exposed to control medium, ET-1 (100 nM) alone or supplemented with AG1478 (1 μ M). Cell lysates were analyzed by IB using anti-active β -cat and anti-HSP70 antibodies. For all panels, data are representative of at least two experiments. Molecular mass is indicated in kilodaltons.

Techniques Used: Immunoprecipitation, Negative Control, Transfection

Related Articles

Small Interfering RNA:

Article Title: Necrotic renal epithelial cell inhibits renal interstitial fibroblast activation: role of protein tyrosine phosphatase 1B
Article Snippet: .. Small interfering RNA (siRNA) specific for rat EGFR and antibodies to fibronectin, EGFR, PTP1B, P2X7, PCNA, and GAPDH were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). α-Smooth muscle actin (α-SMA), α-tubulin, and all other chemicals were purchased from Sigma (St. Louis, MO). .. Phospho tyrosine (PY20) antibody was obtained from BD Biosciences (San Jose, CA).

other:

Article Title: Augmentation of radiation response by panitumumab in models of upper aerodigestive tract cancer
Article Snippet: All other antibodies were purchased from commercial sources as indicated below: EGFR, pEGFR(Tyr1173), and HRP-conjugated goat-anti-rabbit IgG, goat-anti-mouse IgG and donkey-anti-goat IgG antibodies were obtained from Santa Cruz Biotechnology Inc. (Santa Cruz, CA).

Article Title: Enterovirus 71 modulates a COX‐2/PGE2/cAMP‐dependent viral replication in human neuroblastoma cells: Role of the c‐Src/EGFR/p42/p44 MAPK/CREB signaling pathway
Article Snippet: Materials Anti‐COX‐2 (sc‐19999), anti‐p300 (sc‐585), anti‐p44 (sc‐93), anti‐c‐Src (sc‐18), anti‐phospho‐c‐Src (sc‐12928‐R), anti‐EGFR (sc‐03), and anti‐phospho‐EGFR (sc‐23420) were purchased from Santa Cruz (Santa Cruz, CA).

Article Title: Upregulation of sialidase NEU3 in head and neck squamous cell carcinoma associated with lymph node metastasis
Article Snippet: Antibodies Antibodies for phospho‐EGFR (Y‐845), phospho‐ERK, and ERK, from Cell Signaling Technology (Danvers, MA, USA), EGFR from Santa Cruz Biotechnology (Santa Cruz, CA, USA), and a monoclonal anti‐NEU3, prepared as described previously, were used in immunoblotting analysis.

SDS Page:

Article Title: Phosphoproteomics-Based Modeling Defines the Regulatory Mechanism Underlying Aberrant EGFR Signaling
Article Snippet: .. Extracted protein samples were dissolved by SDS-PAGE probed using anti-EGFR and anti-β-tubulin antibodies as a loading control. (0.66 MB TIF) Click here for additional data file. ..

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  • 93
    Santa Cruz Biotechnology total egfr
    Inhibition of cell growth signaling by small molecule inhibitor leads. LNCaP cells were treated with recombinant SEMA3C (0.5 µM) alone or in combination with increasing concentrations of small molecules (0, 2.5, 5, 10, and 20 µM) followed by detection of <t>phospho-EGFR,</t> HER2/ErbB2, SHC, and MAPK. D13 attenuated SEMA3C-induced phosphorylation of EGFR, HER2/ErbB2, SHC, and MAPK. D36 inhibited SEMA3C-induced phosphorylation of MAPK only. D90 attenuated SEMA3C-induced phosphorylation of HER2/ErbB2, p46 SHC, and MAPK. Negative control small molecule <t>D7</t> inhibited phosphorylation of EGFR, HER2/ErbB2, and p46 SHC but only at high concentrations.
    Total Egfr, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 93/100, based on 27 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Santa Cruz Biotechnology anti egfr
    Analysis of the effect of the cellular content on EGF signaling dynamics. A . Experimental validation of relative expression level of model species. Upper right panel: predicted parameter value regarding the initial concentration of model species. Error bars represent standard deviations of 1,000 samples from an ensemble of the WT and Y992F models. Left and lower right panel: western blot analysis of relative concentration of model species. Unstimulated cell lysates of the WT and Y992F cells were dissolved by <t>SDS-PAGE</t> and probed using <t>anti-EGFR,</t> anti-Cbl, anti-Cbl-b, anti-Grb2, anti-Src, anti-Shp2, anti-Shc, anti-Erk1/2, and anti-β-tubulin as a loading control. Quantitated band intensities were normalized to the values for WT. With regard to Shc and Erk proteins, the intensities of three bands of Shc isoforms and two bands of Erk1 and Erk2 were combined, respectively, for the calculation of the relative protein amount. Error bars represent standard deviations of triplicate samples. *P
    Anti Egfr, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 94/100, based on 157 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    89
    Santa Cruz Biotechnology anti egfr af647 antibody
    Four color s-SR imaging using a single fluorophore. (A-B) Four color reconstruction overlay showing clathrin (yellow), α-tubulin (green), actin (orange), and <t>EGFR</t> (blue), imaged sequentially in the order listed, with each target imaged with <t>AF647-conjugated</t> primary antibody with the exception of actin, which was imaged with phalloidin-AF647. (B) Zoomed region highlighted in (A). (C-F) The original reconstruction for each individual component—clathrin (C), α-tubulin (D), actin (E), and EGFR (F)—of the region shown in (B) and highlighted in (A), scale bars 500 nm. Note the lack of any measurable cross-talk between images. (G-J) Two-label reconstructions for clathrin and tubulin (G), tubulin and actin (H), actin and EGFR (I), and clathrin and EGFR (J).
    Anti Egfr Af647 Antibody, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 89/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Santa Cruz Biotechnology epidermal growth factor receptor egfr primary antibodies
    <t>Immunohistochemical</t> studies of (A) caspase-3 and (B) <t>EGFR:</t> (A) (i) normal lung section, (ii)-(iv) BaP-induced lung sections at 5 th , 6 th and 7 th month intervals, respectively, where caspase-3 expression was not found, (v)-(vii) Condurango-30C- treated lung sections at 5 th , 6 th and 7 th month intervals, respectively, where the caspase-3 localization was found, especially at 5 th and 6 th month intervals, indicated by black spots with arrows; (B) (i) normal lung section, (ii)-(iv) BaP induced lung sections at 5 th , 6 th and 7 th month intervals, respectively, where EGFR over-expression was clearly visible (denoted by black spots with arrows), and (v)-(vii) Condurango-30C-treated lung sections at 5 th , 6 th and 7 th month intervals, respectively, where the downregulation of EGFR was found, especially at 5 th and 6 th month time-points.
    Epidermal Growth Factor Receptor Egfr Primary Antibodies, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 86/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Inhibition of cell growth signaling by small molecule inhibitor leads. LNCaP cells were treated with recombinant SEMA3C (0.5 µM) alone or in combination with increasing concentrations of small molecules (0, 2.5, 5, 10, and 20 µM) followed by detection of phospho-EGFR, HER2/ErbB2, SHC, and MAPK. D13 attenuated SEMA3C-induced phosphorylation of EGFR, HER2/ErbB2, SHC, and MAPK. D36 inhibited SEMA3C-induced phosphorylation of MAPK only. D90 attenuated SEMA3C-induced phosphorylation of HER2/ErbB2, p46 SHC, and MAPK. Negative control small molecule D7 inhibited phosphorylation of EGFR, HER2/ErbB2, and p46 SHC but only at high concentrations.

    Journal: Journal of the Endocrine Society

    Article Title: Targeting Semaphorin 3C in Prostate Cancer With Small Molecules

    doi: 10.1210/js.2018-00170

    Figure Lengend Snippet: Inhibition of cell growth signaling by small molecule inhibitor leads. LNCaP cells were treated with recombinant SEMA3C (0.5 µM) alone or in combination with increasing concentrations of small molecules (0, 2.5, 5, 10, and 20 µM) followed by detection of phospho-EGFR, HER2/ErbB2, SHC, and MAPK. D13 attenuated SEMA3C-induced phosphorylation of EGFR, HER2/ErbB2, SHC, and MAPK. D36 inhibited SEMA3C-induced phosphorylation of MAPK only. D90 attenuated SEMA3C-induced phosphorylation of HER2/ErbB2, p46 SHC, and MAPK. Negative control small molecule D7 inhibited phosphorylation of EGFR, HER2/ErbB2, and p46 SHC but only at high concentrations.

    Article Snippet: Notable exceptions were the attenuation of phospho-MAPK by D13 at all concentrations and of phospho-EGFR at higher concentrations of D7 and D90; however, total EGFR also decreased correspondingly in these two treatments [ ].

    Techniques: Inhibition, Recombinant, Negative Control

    Analysis of the effect of the cellular content on EGF signaling dynamics. A . Experimental validation of relative expression level of model species. Upper right panel: predicted parameter value regarding the initial concentration of model species. Error bars represent standard deviations of 1,000 samples from an ensemble of the WT and Y992F models. Left and lower right panel: western blot analysis of relative concentration of model species. Unstimulated cell lysates of the WT and Y992F cells were dissolved by SDS-PAGE and probed using anti-EGFR, anti-Cbl, anti-Cbl-b, anti-Grb2, anti-Src, anti-Shp2, anti-Shc, anti-Erk1/2, and anti-β-tubulin as a loading control. Quantitated band intensities were normalized to the values for WT. With regard to Shc and Erk proteins, the intensities of three bands of Shc isoforms and two bands of Erk1 and Erk2 were combined, respectively, for the calculation of the relative protein amount. Error bars represent standard deviations of triplicate samples. *P

    Journal: PLoS ONE

    Article Title: Phosphoproteomics-Based Modeling Defines the Regulatory Mechanism Underlying Aberrant EGFR Signaling

    doi: 10.1371/journal.pone.0013926

    Figure Lengend Snippet: Analysis of the effect of the cellular content on EGF signaling dynamics. A . Experimental validation of relative expression level of model species. Upper right panel: predicted parameter value regarding the initial concentration of model species. Error bars represent standard deviations of 1,000 samples from an ensemble of the WT and Y992F models. Left and lower right panel: western blot analysis of relative concentration of model species. Unstimulated cell lysates of the WT and Y992F cells were dissolved by SDS-PAGE and probed using anti-EGFR, anti-Cbl, anti-Cbl-b, anti-Grb2, anti-Src, anti-Shp2, anti-Shc, anti-Erk1/2, and anti-β-tubulin as a loading control. Quantitated band intensities were normalized to the values for WT. With regard to Shc and Erk proteins, the intensities of three bands of Shc isoforms and two bands of Erk1 and Erk2 were combined, respectively, for the calculation of the relative protein amount. Error bars represent standard deviations of triplicate samples. *P

    Article Snippet: Extracted protein samples were dissolved by SDS-PAGE probed using anti-EGFR and anti-β-tubulin antibodies as a loading control. (0.66 MB TIF) Click here for additional data file.

    Techniques: Expressing, Concentration Assay, Western Blot, SDS Page

    Four color s-SR imaging using a single fluorophore. (A-B) Four color reconstruction overlay showing clathrin (yellow), α-tubulin (green), actin (orange), and EGFR (blue), imaged sequentially in the order listed, with each target imaged with AF647-conjugated primary antibody with the exception of actin, which was imaged with phalloidin-AF647. (B) Zoomed region highlighted in (A). (C-F) The original reconstruction for each individual component—clathrin (C), α-tubulin (D), actin (E), and EGFR (F)—of the region shown in (B) and highlighted in (A), scale bars 500 nm. Note the lack of any measurable cross-talk between images. (G-J) Two-label reconstructions for clathrin and tubulin (G), tubulin and actin (H), actin and EGFR (I), and clathrin and EGFR (J).

    Journal: PLoS ONE

    Article Title: Sequential Superresolution Imaging of Multiple Targets Using a Single Fluorophore

    doi: 10.1371/journal.pone.0123941

    Figure Lengend Snippet: Four color s-SR imaging using a single fluorophore. (A-B) Four color reconstruction overlay showing clathrin (yellow), α-tubulin (green), actin (orange), and EGFR (blue), imaged sequentially in the order listed, with each target imaged with AF647-conjugated primary antibody with the exception of actin, which was imaged with phalloidin-AF647. (B) Zoomed region highlighted in (A). (C-F) The original reconstruction for each individual component—clathrin (C), α-tubulin (D), actin (E), and EGFR (F)—of the region shown in (B) and highlighted in (A), scale bars 500 nm. Note the lack of any measurable cross-talk between images. (G-J) Two-label reconstructions for clathrin and tubulin (G), tubulin and actin (H), actin and EGFR (I), and clathrin and EGFR (J).

    Article Snippet: Anti-EGFR-AF647 antibody (anti-EGFR, R-1, sc-101) was purchased from Santa Cruz Biotechnology.

    Techniques: Imaging

    Co-localization of EGFR to clathrin-coated vesicles upon activation. HeLa cells were activated with EGF at 1.5 ng/ml for 10 minutes followed by fixation and labeling with anti-EGFR conjugated to AF647. (A) Activation of EGFR results in the formation of puncta in the plasma membrane. (B) Zoomed regions i-iii illustrate the size of EGFR aggregates. (C) After photodestruction, cells were re-labeled with anti-clathrin-AF647 and imaged. The clathrin reconstruction shows ring-like structures in the proximity of EGFR aggregates. (D) The resulting reconstruction overlay (EGFR in green, clathrin in magenta) clearly shows the formation of clathrin-coated vesicles, with clathrin encircling EGFR within these endosomes. We note that neither EGFR aggregates nor any correlation between EGFR and clathrin was observed in the absence of stimulation by EGF (see Fig 4J and S11 Fig ).

    Journal: PLoS ONE

    Article Title: Sequential Superresolution Imaging of Multiple Targets Using a Single Fluorophore

    doi: 10.1371/journal.pone.0123941

    Figure Lengend Snippet: Co-localization of EGFR to clathrin-coated vesicles upon activation. HeLa cells were activated with EGF at 1.5 ng/ml for 10 minutes followed by fixation and labeling with anti-EGFR conjugated to AF647. (A) Activation of EGFR results in the formation of puncta in the plasma membrane. (B) Zoomed regions i-iii illustrate the size of EGFR aggregates. (C) After photodestruction, cells were re-labeled with anti-clathrin-AF647 and imaged. The clathrin reconstruction shows ring-like structures in the proximity of EGFR aggregates. (D) The resulting reconstruction overlay (EGFR in green, clathrin in magenta) clearly shows the formation of clathrin-coated vesicles, with clathrin encircling EGFR within these endosomes. We note that neither EGFR aggregates nor any correlation between EGFR and clathrin was observed in the absence of stimulation by EGF (see Fig 4J and S11 Fig ).

    Article Snippet: Anti-EGFR-AF647 antibody (anti-EGFR, R-1, sc-101) was purchased from Santa Cruz Biotechnology.

    Techniques: Activation Assay, Labeling

    Immunohistochemical studies of (A) caspase-3 and (B) EGFR: (A) (i) normal lung section, (ii)-(iv) BaP-induced lung sections at 5 th , 6 th and 7 th month intervals, respectively, where caspase-3 expression was not found, (v)-(vii) Condurango-30C- treated lung sections at 5 th , 6 th and 7 th month intervals, respectively, where the caspase-3 localization was found, especially at 5 th and 6 th month intervals, indicated by black spots with arrows; (B) (i) normal lung section, (ii)-(iv) BaP induced lung sections at 5 th , 6 th and 7 th month intervals, respectively, where EGFR over-expression was clearly visible (denoted by black spots with arrows), and (v)-(vii) Condurango-30C-treated lung sections at 5 th , 6 th and 7 th month intervals, respectively, where the downregulation of EGFR was found, especially at 5 th and 6 th month time-points.

    Journal: Journal of Pharmacopuncture

    Article Title: Post-cancer Treatment with Condurango 30C Shows Amelioration of Benzo[a]pyrene-induced Lung Cancer in Rats Through the Molecular Pathway of Caspa- se-3-mediated Apoptosis Induction

    doi: 10.3831/KPI.2013.16.021

    Figure Lengend Snippet: Immunohistochemical studies of (A) caspase-3 and (B) EGFR: (A) (i) normal lung section, (ii)-(iv) BaP-induced lung sections at 5 th , 6 th and 7 th month intervals, respectively, where caspase-3 expression was not found, (v)-(vii) Condurango-30C- treated lung sections at 5 th , 6 th and 7 th month intervals, respectively, where the caspase-3 localization was found, especially at 5 th and 6 th month intervals, indicated by black spots with arrows; (B) (i) normal lung section, (ii)-(iv) BaP induced lung sections at 5 th , 6 th and 7 th month intervals, respectively, where EGFR over-expression was clearly visible (denoted by black spots with arrows), and (v)-(vii) Condurango-30C-treated lung sections at 5 th , 6 th and 7 th month intervals, respectively, where the downregulation of EGFR was found, especially at 5 th and 6 th month time-points.

    Article Snippet: Localization of protein distribution by immunohistochemistry and analysis of protein expression by using a Western blot An immunohistochemical study was performed [ ] with caspase-3 and epidermal growth factor receptor (EGFR) primary antibodies and HRP-conjugated secondary antibodies (Santa Cruz Biotechnology, USA).

    Techniques: Immunohistochemistry, Expressing, Over Expression