egfr  (Abcam)

 
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    Name:
    Mouse EGFR peptide
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    Catalog Number:
    ab15739
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    Structured Review

    Abcam egfr
    Silencing of <t>cIAP1</t> reduces <t>EGFR</t> levels. a BT549 and b MCF10A cells were transfected with control or cIAP1-specific siRNAs before being serum-starved overnight, stimulated with 20 ng/ml EGF and analyzed by western blot to detect the indicate proteins. c BT549 cells were transfected with control or cIAP1-specific siRNAs and, after 48 h, cells were serum-starved for 24 h and then stimulated 30 min with 20 ng/ml EGF. Fixed cells were incubated with anti-EGFR antibody and nuclei stained with DAPI. d LRIG1 expression levels were evaluated by real-time PCR in BT549 cells serum-starved and stimulated with 20 ng/ml EGF after transfection with control or cIAP1-directed siRNAs. Unstimulated siCtr vs. sicIAP1 * P = 0.0175, EGF 3 h siCtr vs. sicIAP1 * P = 0.0341; n = 3; two-tailed paired t- test. e Levels of Snai2 and LRIG1 in BT549 cells silenced for cIAP1 and stimulated or not with 20 ng/ml EGF. f EGFR levels in BT549 cells silenced for cIAP1 and LRIG1, stimulated or not with 20 ng/ml EGF. g LRIG1 expression levels were evaluated by real-time PCR in MCF10A cells serum-starved and stimulated with 20 ng/ml EGF after transfection with control or cIAP1-directed siRNAs. ns not significant; n = 4; two-tailed paired t- test

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    Images

    1) Product Images from "cIAP1 regulates the EGFR/Snai2 axis in triple-negative breast cancer cells"

    Article Title: cIAP1 regulates the EGFR/Snai2 axis in triple-negative breast cancer cells

    Journal: Cell Death and Differentiation

    doi: 10.1038/s41418-018-0100-0

    Silencing of cIAP1 reduces EGFR levels. a BT549 and b MCF10A cells were transfected with control or cIAP1-specific siRNAs before being serum-starved overnight, stimulated with 20 ng/ml EGF and analyzed by western blot to detect the indicate proteins. c BT549 cells were transfected with control or cIAP1-specific siRNAs and, after 48 h, cells were serum-starved for 24 h and then stimulated 30 min with 20 ng/ml EGF. Fixed cells were incubated with anti-EGFR antibody and nuclei stained with DAPI. d LRIG1 expression levels were evaluated by real-time PCR in BT549 cells serum-starved and stimulated with 20 ng/ml EGF after transfection with control or cIAP1-directed siRNAs. Unstimulated siCtr vs. sicIAP1 * P = 0.0175, EGF 3 h siCtr vs. sicIAP1 * P = 0.0341; n = 3; two-tailed paired t- test. e Levels of Snai2 and LRIG1 in BT549 cells silenced for cIAP1 and stimulated or not with 20 ng/ml EGF. f EGFR levels in BT549 cells silenced for cIAP1 and LRIG1, stimulated or not with 20 ng/ml EGF. g LRIG1 expression levels were evaluated by real-time PCR in MCF10A cells serum-starved and stimulated with 20 ng/ml EGF after transfection with control or cIAP1-directed siRNAs. ns not significant; n = 4; two-tailed paired t- test
    Figure Legend Snippet: Silencing of cIAP1 reduces EGFR levels. a BT549 and b MCF10A cells were transfected with control or cIAP1-specific siRNAs before being serum-starved overnight, stimulated with 20 ng/ml EGF and analyzed by western blot to detect the indicate proteins. c BT549 cells were transfected with control or cIAP1-specific siRNAs and, after 48 h, cells were serum-starved for 24 h and then stimulated 30 min with 20 ng/ml EGF. Fixed cells were incubated with anti-EGFR antibody and nuclei stained with DAPI. d LRIG1 expression levels were evaluated by real-time PCR in BT549 cells serum-starved and stimulated with 20 ng/ml EGF after transfection with control or cIAP1-directed siRNAs. Unstimulated siCtr vs. sicIAP1 * P = 0.0175, EGF 3 h siCtr vs. sicIAP1 * P = 0.0341; n = 3; two-tailed paired t- test. e Levels of Snai2 and LRIG1 in BT549 cells silenced for cIAP1 and stimulated or not with 20 ng/ml EGF. f EGFR levels in BT549 cells silenced for cIAP1 and LRIG1, stimulated or not with 20 ng/ml EGF. g LRIG1 expression levels were evaluated by real-time PCR in MCF10A cells serum-starved and stimulated with 20 ng/ml EGF after transfection with control or cIAP1-directed siRNAs. ns not significant; n = 4; two-tailed paired t- test

    Techniques Used: Transfection, Western Blot, Incubation, Staining, Expressing, Real-time Polymerase Chain Reaction, Two Tailed Test

    SM83 treatment results in anti-tumor and anti-metastasis effect. a for primary tumor volumes), formalin-fixed and paraffin-embedded, and stained with an anti-human vimentin antibody to detect spontaneous metastasis (bottom panel). b Number (untreated n = 7, SM83-treated mice n ; * P = 0.0238. Unpaired two-tailed t- test) and c size (35 metastases/group; * P = 0.0107. Unpaired two-tailed t- test) of spontaneous MDA-MB231 lung metastases were evaluated. d Western blot showing the levels of cIAP1, EGFR, ERK1/2, pERK1/2, and Snai2 in primary tumors at the end of the experiment described in Fig. 8a
    Figure Legend Snippet: SM83 treatment results in anti-tumor and anti-metastasis effect. a for primary tumor volumes), formalin-fixed and paraffin-embedded, and stained with an anti-human vimentin antibody to detect spontaneous metastasis (bottom panel). b Number (untreated n = 7, SM83-treated mice n ; * P = 0.0238. Unpaired two-tailed t- test) and c size (35 metastases/group; * P = 0.0107. Unpaired two-tailed t- test) of spontaneous MDA-MB231 lung metastases were evaluated. d Western blot showing the levels of cIAP1, EGFR, ERK1/2, pERK1/2, and Snai2 in primary tumors at the end of the experiment described in Fig. 8a

    Techniques Used: Staining, Mouse Assay, Two Tailed Test, Multiple Displacement Amplification, Western Blot

    cIAP1 reduces EGFR stability, but promotes its gene transcription. a EGFR and cIAP1 interaction was tested in BT549 cells using PLA assay. b BT549 cells stably expressing Myc/Flag-tagged EGFR were serum-starved and stimulated with 20 ng/ml EGF for the indicated times. Cells were lysed and EGFR was immunoprecipitated with anti-Flag antibody. Western blot was performed to evaluate the interaction of ectopic EGFR with cIAP1 and c-Cbl. c BT549 and MCF10A cells stably expressing Myc/Flag-tagged EGFR were serum-starved, pre-treated with 100 (BT549) or 50 (MCF10A) µg/ml cycloheximide for 30 min and stimulated with 20 ng/ml EGF for the indicated times. Levels of ectopic EGFR were detected with anti-Myc antibody. d Ectopic EGFR was immunoprecipitated as described in Fig. 6b from BT549 cells transfected with control and cIAP1-specific siRNAs. Western blot shows total levels of c-Cbl and the amount of c-Cbl interacting with EGFR. e BT549 and f MCF10A cell stably expressing ectopic Myc/Flag-tagged EGFR were further transduced with lentiviral particles to overexpress c-Cbl or GFP as a control. Cells were silenced for cIAP1, serum-starved, and stimulated with 20 ng/ml EGF as described above, and analyzed by western blot to evaluate the levels of ectopic EGFR by using a Myc-tagged-specific antibody. g BT549 (left panel) and MCF10A (right panel) were silenced for cIAP1 and analyzed by real-time PCR to quantify the levels of EGFR expression fold relative to GAPDH. BT549: unstimulated siCtr vs. sicIAP1 * P = 0.0134, EGF 3 h siCtr vs. sicIAP1 * P = 0.0270; n = 3. MCF10A: unstimulated siCtr vs. sicIAP1 *** P = 0.0004, EGF 3 h siCtr vs. sicIAP1 ** P = 0.0183; n = 4; two-tailed paired t- test
    Figure Legend Snippet: cIAP1 reduces EGFR stability, but promotes its gene transcription. a EGFR and cIAP1 interaction was tested in BT549 cells using PLA assay. b BT549 cells stably expressing Myc/Flag-tagged EGFR were serum-starved and stimulated with 20 ng/ml EGF for the indicated times. Cells were lysed and EGFR was immunoprecipitated with anti-Flag antibody. Western blot was performed to evaluate the interaction of ectopic EGFR with cIAP1 and c-Cbl. c BT549 and MCF10A cells stably expressing Myc/Flag-tagged EGFR were serum-starved, pre-treated with 100 (BT549) or 50 (MCF10A) µg/ml cycloheximide for 30 min and stimulated with 20 ng/ml EGF for the indicated times. Levels of ectopic EGFR were detected with anti-Myc antibody. d Ectopic EGFR was immunoprecipitated as described in Fig. 6b from BT549 cells transfected with control and cIAP1-specific siRNAs. Western blot shows total levels of c-Cbl and the amount of c-Cbl interacting with EGFR. e BT549 and f MCF10A cell stably expressing ectopic Myc/Flag-tagged EGFR were further transduced with lentiviral particles to overexpress c-Cbl or GFP as a control. Cells were silenced for cIAP1, serum-starved, and stimulated with 20 ng/ml EGF as described above, and analyzed by western blot to evaluate the levels of ectopic EGFR by using a Myc-tagged-specific antibody. g BT549 (left panel) and MCF10A (right panel) were silenced for cIAP1 and analyzed by real-time PCR to quantify the levels of EGFR expression fold relative to GAPDH. BT549: unstimulated siCtr vs. sicIAP1 * P = 0.0134, EGF 3 h siCtr vs. sicIAP1 * P = 0.0270; n = 3. MCF10A: unstimulated siCtr vs. sicIAP1 *** P = 0.0004, EGF 3 h siCtr vs. sicIAP1 ** P = 0.0183; n = 4; two-tailed paired t- test

    Techniques Used: Proximity Ligation Assay, Stable Transfection, Expressing, Immunoprecipitation, Western Blot, Transfection, Transduction, Real-time Polymerase Chain Reaction, Two Tailed Test

    Depletion of cIAP1 hinders EGFR-dependent expression of Snai2. a MDA-MB231 and b BT549 cells were transfected with control or cIAP1-specific siRNAs and, after 48 h, serum-starved overnight. Then, cells were stimulated for the indicated time points with 50 ng/ml and 20 ng/ml EGF, respectively. Levels of Snai2 and activated ERK1/2 were detected, together with cIAP1, to check the transfection efficiency. c BT549 and d MCF10A—wild-type or bearing mutated EGFR—cells were transfected as in Fig. 4a and stimulated with the indicated EGFR ligands (20 ng/ml) to evaluate the expression of Snai2 by western blot. e BT549 and f MCF10A cells were transfected and serum-starved as described before, stimulated 3 h with 20 ng/ml EGF and lysed to extract RNA. Real-time PCR was performed to evaluate Snai2 fold expression relative to GAPDH. BT549: * P = 0.0151, ** P = 0.0036; n = 3; MCF10A: unstimulated siCtr vs. sicIAP1 * P = 0.0290, EGF 3 h siCtr vs. sicIAP1 * P = 0.0330; n = 5; two-tailed paired t- test
    Figure Legend Snippet: Depletion of cIAP1 hinders EGFR-dependent expression of Snai2. a MDA-MB231 and b BT549 cells were transfected with control or cIAP1-specific siRNAs and, after 48 h, serum-starved overnight. Then, cells were stimulated for the indicated time points with 50 ng/ml and 20 ng/ml EGF, respectively. Levels of Snai2 and activated ERK1/2 were detected, together with cIAP1, to check the transfection efficiency. c BT549 and d MCF10A—wild-type or bearing mutated EGFR—cells were transfected as in Fig. 4a and stimulated with the indicated EGFR ligands (20 ng/ml) to evaluate the expression of Snai2 by western blot. e BT549 and f MCF10A cells were transfected and serum-starved as described before, stimulated 3 h with 20 ng/ml EGF and lysed to extract RNA. Real-time PCR was performed to evaluate Snai2 fold expression relative to GAPDH. BT549: * P = 0.0151, ** P = 0.0036; n = 3; MCF10A: unstimulated siCtr vs. sicIAP1 * P = 0.0290, EGF 3 h siCtr vs. sicIAP1 * P = 0.0330; n = 5; two-tailed paired t- test

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

    EGFR promotes Snai2 expression in a MAPK-dependent manner. a MDA-MB231, BT549, and MDA-MB157 cells were harvested after treatment for 2 h with 10 μM inhibitor of PI3K (LY294002), AKT (Triciribine), MEK (UO126), and p38 (SB203580). Western blot was performed to analyze the levels of Snai2. b MDA-MB231 cells were transfected with siRNAs specific for cIAP1, ERK1, and ERK2 to detect the levels of Snai2 72 h after transfection. cIAP1 and ERK1/2 are shown to control the silencing efficiency. c ]. d For each cancer type available in the TCGA study, Spearman’s correlation between EGFR and SNAI2 was calculated using RNA-Seq data (expressed as log2 counts per million mapped reads). Only primary tumors were considered in the analysis. Red arrow indicates the correlation bar in breast cancers. e BT549 cells were serum-starved overnight and then stimulated with 20 ng/ml EGF and TGFα in time-course experiments. Snai2 levels are shown together with total and activated levels of EGFR. MDA-MB231 ( f ) and BT549 ( g ) cells were serum-starved overnight, pre-treated or not with 100 μg/ml cetuximab for 1 h and then stimulated with 20 ng/ml EGF for the indicated time points. Western blot was performed to detect Snai2 levels, total ERK1/2 and EGFR, and their activated levels. Values show the fold levels of Snai2 relative to untreated cells. h Human mammary epithelial cell lines, parental and bearing mutated EGFR, were serum-starved and stimulated with 20 ng/ml EGF for the indicated times to evaluate Snai2 levels
    Figure Legend Snippet: EGFR promotes Snai2 expression in a MAPK-dependent manner. a MDA-MB231, BT549, and MDA-MB157 cells were harvested after treatment for 2 h with 10 μM inhibitor of PI3K (LY294002), AKT (Triciribine), MEK (UO126), and p38 (SB203580). Western blot was performed to analyze the levels of Snai2. b MDA-MB231 cells were transfected with siRNAs specific for cIAP1, ERK1, and ERK2 to detect the levels of Snai2 72 h after transfection. cIAP1 and ERK1/2 are shown to control the silencing efficiency. c ]. d For each cancer type available in the TCGA study, Spearman’s correlation between EGFR and SNAI2 was calculated using RNA-Seq data (expressed as log2 counts per million mapped reads). Only primary tumors were considered in the analysis. Red arrow indicates the correlation bar in breast cancers. e BT549 cells were serum-starved overnight and then stimulated with 20 ng/ml EGF and TGFα in time-course experiments. Snai2 levels are shown together with total and activated levels of EGFR. MDA-MB231 ( f ) and BT549 ( g ) cells were serum-starved overnight, pre-treated or not with 100 μg/ml cetuximab for 1 h and then stimulated with 20 ng/ml EGF for the indicated time points. Western blot was performed to detect Snai2 levels, total ERK1/2 and EGFR, and their activated levels. Values show the fold levels of Snai2 relative to untreated cells. h Human mammary epithelial cell lines, parental and bearing mutated EGFR, were serum-starved and stimulated with 20 ng/ml EGF for the indicated times to evaluate Snai2 levels

    Techniques Used: Expressing, Multiple Displacement Amplification, Western Blot, Transfection, RNA Sequencing Assay

    2) Product Images from "Radiotherapy Upregulates Programmed Death Ligand-1 through the Pathways Downstream of Epidermal Growth Factor Receptor in Glioma"

    Article Title: Radiotherapy Upregulates Programmed Death Ligand-1 through the Pathways Downstream of Epidermal Growth Factor Receptor in Glioma

    Journal: EBioMedicine

    doi: 10.1016/j.ebiom.2018.01.027

    RT activates the EGFR signaling and up-regulates the PD-L1 expression in glioma cells. The EGFR signaling and PD-L1 protein level in U87 and U251 cell lines were detected at 24, 48 and 72 h after 5 or 10 Gy irradiation. Representative images and quantitative data are shown. Each column is shown as the means of three separate experiments; bars, SD. ANOVA; * P
    Figure Legend Snippet: RT activates the EGFR signaling and up-regulates the PD-L1 expression in glioma cells. The EGFR signaling and PD-L1 protein level in U87 and U251 cell lines were detected at 24, 48 and 72 h after 5 or 10 Gy irradiation. Representative images and quantitative data are shown. Each column is shown as the means of three separate experiments; bars, SD. ANOVA; * P

    Techniques Used: Expressing, Irradiation

    EGFR inhibitor abrogates the up-regulation of PD-L1 at the mRNA level caused by RT in glioma cells. Cell lines (U87 and U251) were treated with vehicle control, RT (5 or 10 Gy), or RT combined with AG490 (10 μM) for 24, 48 or 72 h. PD-L1 expression at the mRNA level was determined by RT-PCR and expressed as fold change relative to the vehicle group. Each column is shown as the means of three separate experiments; bars, SD. ANOVA; *** P
    Figure Legend Snippet: EGFR inhibitor abrogates the up-regulation of PD-L1 at the mRNA level caused by RT in glioma cells. Cell lines (U87 and U251) were treated with vehicle control, RT (5 or 10 Gy), or RT combined with AG490 (10 μM) for 24, 48 or 72 h. PD-L1 expression at the mRNA level was determined by RT-PCR and expressed as fold change relative to the vehicle group. Each column is shown as the means of three separate experiments; bars, SD. ANOVA; *** P

    Techniques Used: Expressing, Reverse Transcription Polymerase Chain Reaction

    EGFR inhibitor abrogates the up-regulation of PD-L1 caused by RT in glioma cells. EGFR signaling and PD-L1 protein level in U87 and U251 cell lines were detected at 24, 48 and 72 h after 5 or 10 Gy irradiation. Before irradiation, U87 and U251 cells were incubated in the presence of 10 μM AG490 at 37 °C for 1 h. Each column is shown as the means of three separate experiments; bars, SD. ANOVA; * P
    Figure Legend Snippet: EGFR inhibitor abrogates the up-regulation of PD-L1 caused by RT in glioma cells. EGFR signaling and PD-L1 protein level in U87 and U251 cell lines were detected at 24, 48 and 72 h after 5 or 10 Gy irradiation. Before irradiation, U87 and U251 cells were incubated in the presence of 10 μM AG490 at 37 °C for 1 h. Each column is shown as the means of three separate experiments; bars, SD. ANOVA; * P

    Techniques Used: Irradiation, Incubation

    3) 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

    Synergic effect of 4D3 anti-CLDN4 antibody with anti-EGFR antibody ( A ) Effect of 4D3 on growth inhibition induced by anti-EGFR antibody C225 in spheres of Caco-2 cells. Anti-mouse CLDN4 was treated as a negative control. ( B ) Permeation of Caco-2 cell spheres with antibodies. Spheres were treated with fluorescence-labeled 4D3 and C225. Nuclei were stained by DAPI. ( C ) Effect of co-treatment with 4D3 and C225 on phosphorylation of EGFR in Caco-2 cell spheres. ( D , E ) Effect of 4D3 on C225-induced inhibition of tumor growth in nude mice ( n = 5). ( F ) Effect of time interval (h) between treatment with 4D3 and treatment with C225 on phosphorylation of EGFR in spheres. ( G ) Effects of sequential (Seq, 6-h time interval) vs. synchronous (Syn) treatment with 4D3 and C225 on tumor growth of Caco-2 cells in nude mice ( n = 5).
    Figure Legend Snippet: Synergic effect of 4D3 anti-CLDN4 antibody with anti-EGFR antibody ( A ) Effect of 4D3 on growth inhibition induced by anti-EGFR antibody C225 in spheres of Caco-2 cells. Anti-mouse CLDN4 was treated as a negative control. ( B ) Permeation of Caco-2 cell spheres with antibodies. Spheres were treated with fluorescence-labeled 4D3 and C225. Nuclei were stained by DAPI. ( C ) Effect of co-treatment with 4D3 and C225 on phosphorylation of EGFR in Caco-2 cell spheres. ( D , E ) Effect of 4D3 on C225-induced inhibition of tumor growth in nude mice ( n = 5). ( F ) Effect of time interval (h) between treatment with 4D3 and treatment with C225 on phosphorylation of EGFR in spheres. ( G ) Effects of sequential (Seq, 6-h time interval) vs. synchronous (Syn) treatment with 4D3 and C225 on tumor growth of Caco-2 cells in nude mice ( n = 5).

    Techniques Used: Inhibition, Negative Control, Fluorescence, Labeling, Staining, Mouse Assay

    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

    4) Product Images from "ING5 knockdown enhances migration and invasion of lung cancer cells by inducing EMT via EGFR/PI3K/Akt and IL-6/STAT3 signaling pathways"

    Article Title: ING5 knockdown enhances migration and invasion of lung cancer cells by inducing EMT via EGFR/PI3K/Akt and IL-6/STAT3 signaling pathways

    Journal: Oncotarget

    doi: 10.18632/oncotarget.17346

    ING5 knockdown activates EGFR/PI3K/Akt and IL-6/STAT3 signaling pathways ( A ) The Phospho-Kinase antibody array was incubated with cell lysates from shControl and shING5 A549 cells. Data shown are from a 2 minute exposures to film. ( B ) Twelve pairs of duplicate spots representing 12 proteins were selected and the average signal density was calculated. The relative change of protein phosphorylation upon ING5 knockdown was determined by the ratio of corresponding signals from shING5 to shControl array film. 1. PRAS40 (T246); 2. ERK1/2 (T202/Y204, T185/Y187); 3. STAT2 (Y689); 4. Akt1/2/3 (S473); 5. STAT5b (Y699); 6. STAT5a/b (Y694/Y699); 7. Akt1/2/3 (T308); 8. P70S6 Kinase (T421/S424); 9. STAT3 (Y705); 10. P53 (S392); 11. P53 (S46); 12. P53 (S15). ( C ) Effects of ING5 overexpression or knockdown on protein expression involved in PI3K and STAT3 pathways. Protein level of total Akt, P-Akt S473, P-Akt T308, total STAT3, P-STAT3 Y705, EGFR and IL-6 were detected by western blot. Actin was used as an internal loading control.
    Figure Legend Snippet: ING5 knockdown activates EGFR/PI3K/Akt and IL-6/STAT3 signaling pathways ( A ) The Phospho-Kinase antibody array was incubated with cell lysates from shControl and shING5 A549 cells. Data shown are from a 2 minute exposures to film. ( B ) Twelve pairs of duplicate spots representing 12 proteins were selected and the average signal density was calculated. The relative change of protein phosphorylation upon ING5 knockdown was determined by the ratio of corresponding signals from shING5 to shControl array film. 1. PRAS40 (T246); 2. ERK1/2 (T202/Y204, T185/Y187); 3. STAT2 (Y689); 4. Akt1/2/3 (S473); 5. STAT5b (Y699); 6. STAT5a/b (Y694/Y699); 7. Akt1/2/3 (T308); 8. P70S6 Kinase (T421/S424); 9. STAT3 (Y705); 10. P53 (S392); 11. P53 (S46); 12. P53 (S15). ( C ) Effects of ING5 overexpression or knockdown on protein expression involved in PI3K and STAT3 pathways. Protein level of total Akt, P-Akt S473, P-Akt T308, total STAT3, P-STAT3 Y705, EGFR and IL-6 were detected by western blot. Actin was used as an internal loading control.

    Techniques Used: Ab Array, Incubation, Over Expression, Expressing, Western Blot

    Inhibition of STAT3 or PI3K/Akt pathway reverses ING5 knockdown-induced EMT ( A ) Effects of Niclosamide or ZSTK474 on expression of proteins involved in EGFR/PI3K/Akt and IL-6/STAT3 pathways by western blot. Actin was used as an internal loading control. ( B ) Effects of Niclosamide or ZSTK474 on the expression of E-cadherin by immunofluorescence staining in shControl and shING5 A549 cells. Representative pictures are shown (200× magnification). Data are shown as mean plus standard error of at least three independent experiments. The MFI value (mean fluorescence intensity) was compared. ** P
    Figure Legend Snippet: Inhibition of STAT3 or PI3K/Akt pathway reverses ING5 knockdown-induced EMT ( A ) Effects of Niclosamide or ZSTK474 on expression of proteins involved in EGFR/PI3K/Akt and IL-6/STAT3 pathways by western blot. Actin was used as an internal loading control. ( B ) Effects of Niclosamide or ZSTK474 on the expression of E-cadherin by immunofluorescence staining in shControl and shING5 A549 cells. Representative pictures are shown (200× magnification). Data are shown as mean plus standard error of at least three independent experiments. The MFI value (mean fluorescence intensity) was compared. ** P

    Techniques Used: Inhibition, Expressing, Western Blot, Immunofluorescence, Staining, Fluorescence

    5) Product Images from "BRCA1-IRIS inactivation overcomes paclitaxel resistance in triple negative breast cancers"

    Article Title: BRCA1-IRIS inactivation overcomes paclitaxel resistance in triple negative breast cancers

    Journal: Breast Cancer Research : BCR

    doi: 10.1186/s13058-014-0512-9

    BRCA1-IRIS overexpression promotes intrinsic and acquired paclitaxel resistance in TNBC cells. (A) The survival of HME, HME/IRIS and the indicated TNBC cell lines following treatment with increasing concentrations of paclitaxel. Values are means of triplicates done three separate times. Inset shows BRCA1-IRIS expression in these cell lines following exposure to increasing concentrations of paclitaxel. (B) The expression of the indicated proteins following treatment with vehicle or paclitaxel (5 μM) for 24 h or MDA-MB-468 previously silenced from luciferase or BRCA1-IRIS for 48 h. (C) The expression of the indicated proteins in HME or HME/IRIS cells following exposure to 0, 10, 20 or 30 μM of paclitaxel. (D) The expression of the indicated proteins in HME or HME/IRIS cells following exposure to 1 μM of paclitaxel for 0, 1 or 3 weeks. (E and F) The expression of the indicated proteins in the nucleus or cytoplasm of HME or HME/IRIS following exposure to 1 μM of paclitaxel for 0, 1 or 3 weeks. Activated ERK, JNK and p38 were detected using antibodies specifically detect p- T202/Y204 -ERK, p- T183/Y185 -JNK, and p- T180/Y182 -p38. (G) The effect of BRCA1-IRIS overexpression on the proliferation of HME cells. (H) The effect of inhibiting ERK (using PD98059), JNK (using SP600125), p38 (using SB203580), PI3′K/AKT (using LY294002), EGFR (using Erlotinib), ErbB2 (using CP-724714), EGFR/ErbB2 (using Lapatinib), and EGFR/ErbB2/ErbB3 (using Sapitinib) on the survival of HME or HME/IRIS cells. Values represent the means of experiments that were performed in triplicate done three separate times, *** = P ≤0.001 (compared to control in each cell line) . (I) Schematic representation of the data so far. EGFR, ErbB2 and ErbB3, epidermal growth factor receptor 1, 2 and 3, respectively; HME, human mammary epithelial cells; TNBC, triple negative breast cancer.
    Figure Legend Snippet: BRCA1-IRIS overexpression promotes intrinsic and acquired paclitaxel resistance in TNBC cells. (A) The survival of HME, HME/IRIS and the indicated TNBC cell lines following treatment with increasing concentrations of paclitaxel. Values are means of triplicates done three separate times. Inset shows BRCA1-IRIS expression in these cell lines following exposure to increasing concentrations of paclitaxel. (B) The expression of the indicated proteins following treatment with vehicle or paclitaxel (5 μM) for 24 h or MDA-MB-468 previously silenced from luciferase or BRCA1-IRIS for 48 h. (C) The expression of the indicated proteins in HME or HME/IRIS cells following exposure to 0, 10, 20 or 30 μM of paclitaxel. (D) The expression of the indicated proteins in HME or HME/IRIS cells following exposure to 1 μM of paclitaxel for 0, 1 or 3 weeks. (E and F) The expression of the indicated proteins in the nucleus or cytoplasm of HME or HME/IRIS following exposure to 1 μM of paclitaxel for 0, 1 or 3 weeks. Activated ERK, JNK and p38 were detected using antibodies specifically detect p- T202/Y204 -ERK, p- T183/Y185 -JNK, and p- T180/Y182 -p38. (G) The effect of BRCA1-IRIS overexpression on the proliferation of HME cells. (H) The effect of inhibiting ERK (using PD98059), JNK (using SP600125), p38 (using SB203580), PI3′K/AKT (using LY294002), EGFR (using Erlotinib), ErbB2 (using CP-724714), EGFR/ErbB2 (using Lapatinib), and EGFR/ErbB2/ErbB3 (using Sapitinib) on the survival of HME or HME/IRIS cells. Values represent the means of experiments that were performed in triplicate done three separate times, *** = P ≤0.001 (compared to control in each cell line) . (I) Schematic representation of the data so far. EGFR, ErbB2 and ErbB3, epidermal growth factor receptor 1, 2 and 3, respectively; HME, human mammary epithelial cells; TNBC, triple negative breast cancer.

    Techniques Used: Over Expression, Expressing, Multiple Displacement Amplification, Luciferase

    Elevated BRCA1-IRIS and survivin and lack of FOXO3a expression correlates with breast tumors aggressiveness. Paraffin-embedded tissue microarray sections were examined by immunohistochemistry with anti-BRCA1-IRIS, survivin and FOXO3a mAb. (A and B) BRCA1-IRIS and survivin, respectively staining scores in normal (n = 66), DCIS (n = 167), invasive (n = 179) and metastatic (n = 99) breast cancer tissue samples. (C and D) BRCA1-IRIS and survivin, respectively staining scores per field as compared to the localization of FOXO3a in each cell in normal (n = 66), DCIS (n = 167), invasive (n = 179) and metastatic (n = 99) breast cancer tissue samples. (E) Percentage of probability of disease-free survival in patients with TNBC tumors overexpressing low (blue line) vs. middle/high (red line) levels of EGFR/AKT/MDM2/Skp2/survivin as a surrogate for BRCA-IRIS overexpression. DCIS, ductal carcinoma in situ ; FOXO3a, Forkhead box class OAKT3a; mAb, monoclonal antibody; TNBC, triple negative breast cancer.
    Figure Legend Snippet: Elevated BRCA1-IRIS and survivin and lack of FOXO3a expression correlates with breast tumors aggressiveness. Paraffin-embedded tissue microarray sections were examined by immunohistochemistry with anti-BRCA1-IRIS, survivin and FOXO3a mAb. (A and B) BRCA1-IRIS and survivin, respectively staining scores in normal (n = 66), DCIS (n = 167), invasive (n = 179) and metastatic (n = 99) breast cancer tissue samples. (C and D) BRCA1-IRIS and survivin, respectively staining scores per field as compared to the localization of FOXO3a in each cell in normal (n = 66), DCIS (n = 167), invasive (n = 179) and metastatic (n = 99) breast cancer tissue samples. (E) Percentage of probability of disease-free survival in patients with TNBC tumors overexpressing low (blue line) vs. middle/high (red line) levels of EGFR/AKT/MDM2/Skp2/survivin as a surrogate for BRCA-IRIS overexpression. DCIS, ductal carcinoma in situ ; FOXO3a, Forkhead box class OAKT3a; mAb, monoclonal antibody; TNBC, triple negative breast cancer.

    Techniques Used: Expressing, Microarray, Immunohistochemistry, Staining, Over Expression, In Situ

    6) Product Images from "A CD44high/EGFRlow Subpopulation within Head and Neck Cancer Cell Lines Shows an Epithelial-Mesenchymal Transition Phenotype and Resistance to Treatment"

    Article Title: A CD44high/EGFRlow Subpopulation within Head and Neck Cancer Cell Lines Shows an Epithelial-Mesenchymal Transition Phenotype and Resistance to Treatment

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0044071

    The CD44 high population shows upregulation of EMT-associated genes. A–C: Relative mRNA expression levels of epithelial-mesenchymal transition-associated genes; E-cadherin, N-cadherin, vimentin, fibronectin1, Twist1, FOXC2 and matrix metalloproteinase 7 (MMP7) in ( A ) LK0923, ( B ) LK0827 and ( C ) LK0863 subpopulations. The expression levels in the CD44 high /EGFR high and CD44 high /EGFR low populations are displayed here as fold difference relative to the CD44 low population (GAPDH was used as an internal standard). D: Western blot analysis of N-cadherin, vimentin and fibronectin1 in LK0923 populations (β-actin was used as loading control).
    Figure Legend Snippet: The CD44 high population shows upregulation of EMT-associated genes. A–C: Relative mRNA expression levels of epithelial-mesenchymal transition-associated genes; E-cadherin, N-cadherin, vimentin, fibronectin1, Twist1, FOXC2 and matrix metalloproteinase 7 (MMP7) in ( A ) LK0923, ( B ) LK0827 and ( C ) LK0863 subpopulations. The expression levels in the CD44 high /EGFR high and CD44 high /EGFR low populations are displayed here as fold difference relative to the CD44 low population (GAPDH was used as an internal standard). D: Western blot analysis of N-cadherin, vimentin and fibronectin1 in LK0923 populations (β-actin was used as loading control).

    Techniques Used: Expressing, Western Blot

    Sorted subpopulations show differences in treatment sensitivity. Treatment sensitivity of CD44 low , CD44 high /EGFR high and CD44 high /EGFR low cells, isolated from ( A ) LK0923, ( B ) LK0827 or ( C ) LK0863, and exposed to radiation (4 Gy), cisplatin (2 μg/ml, 1h), cetuximab (30 nM), gefitinib (50 nM) or dasatinib (10 nM). Data represents mean ± SD from three experiments in triplicate, *p≤0.05.
    Figure Legend Snippet: Sorted subpopulations show differences in treatment sensitivity. Treatment sensitivity of CD44 low , CD44 high /EGFR high and CD44 high /EGFR low cells, isolated from ( A ) LK0923, ( B ) LK0827 or ( C ) LK0863, and exposed to radiation (4 Gy), cisplatin (2 μg/ml, 1h), cetuximab (30 nM), gefitinib (50 nM) or dasatinib (10 nM). Data represents mean ± SD from three experiments in triplicate, *p≤0.05.

    Techniques Used: Isolation

    The CD44 high /EGFR low population shows upregulation of cancer stem cell-associated genes. Relative mRNA expression levels of the stemness genes NANOG and SOX1 in LK0923, LK0827 and LK0863 subpopulations, here displayed as fold difference relative to the CD44 low population (GAPDH was used as an internal standard).
    Figure Legend Snippet: The CD44 high /EGFR low population shows upregulation of cancer stem cell-associated genes. Relative mRNA expression levels of the stemness genes NANOG and SOX1 in LK0923, LK0827 and LK0863 subpopulations, here displayed as fold difference relative to the CD44 low population (GAPDH was used as an internal standard).

    Techniques Used: Expressing

    A: Identification of subpopulations of cells in HNSCC cell lines. Dot plot analysis of LK0923, LK0827 and LK0863 cells co-stained with an APC-conjugated anti-CD44 antibody and a PE-conjugated anti-epidermal growth factor receptor (EGFR) antibody. The red colored areas represent the CD44 high cells, and the three boxes display the gating of the different subpopulations from which cells were isolated for further experiments. B: Light microscope images of CD44 low , CD44 high /EGFR high and CD44 high /EGFR low populations (3 days after sorting) and unsorted LK0923, LK0827 and LK0863 cell cultures.
    Figure Legend Snippet: A: Identification of subpopulations of cells in HNSCC cell lines. Dot plot analysis of LK0923, LK0827 and LK0863 cells co-stained with an APC-conjugated anti-CD44 antibody and a PE-conjugated anti-epidermal growth factor receptor (EGFR) antibody. The red colored areas represent the CD44 high cells, and the three boxes display the gating of the different subpopulations from which cells were isolated for further experiments. B: Light microscope images of CD44 low , CD44 high /EGFR high and CD44 high /EGFR low populations (3 days after sorting) and unsorted LK0923, LK0827 and LK0863 cell cultures.

    Techniques Used: Staining, Isolation, Light Microscopy

    Characterization of the CD44 low , CD44 high /EGFR high and CD44 high /EGFR low subpopulations within the LK0923 cell line. A: Expression of CD44 and epidermal growth factor receptor (EGFR) in sorted subpopulations (β-actin was used as a loading control). B: Proliferation rate of subpopulations. Data represents mean ± SD from three experiments in triplicate, *p≤0.05. C: Cell cycle distribution comparison between CD44 low and CD44 high /EGFR low cells. Data from one representative experiment out of two are shown. D: Plating efficiency, as assessed by colony formation in sorted subpopulations. Data represents mean ± SD from three experiments in duplicate, *p≤0.05.
    Figure Legend Snippet: Characterization of the CD44 low , CD44 high /EGFR high and CD44 high /EGFR low subpopulations within the LK0923 cell line. A: Expression of CD44 and epidermal growth factor receptor (EGFR) in sorted subpopulations (β-actin was used as a loading control). B: Proliferation rate of subpopulations. Data represents mean ± SD from three experiments in triplicate, *p≤0.05. C: Cell cycle distribution comparison between CD44 low and CD44 high /EGFR low cells. Data from one representative experiment out of two are shown. D: Plating efficiency, as assessed by colony formation in sorted subpopulations. Data represents mean ± SD from three experiments in duplicate, *p≤0.05.

    Techniques Used: Expressing

    7) Product Images from "Dihydrotestosterone Potentiates EGF-Induced ERK Activation by Inducing SRC in Fetal Lung Fibroblasts"

    Article Title: Dihydrotestosterone Potentiates EGF-Induced ERK Activation by Inducing SRC in Fetal Lung Fibroblasts

    Journal: American Journal of Respiratory Cell and Molecular Biology

    doi: 10.1165/rcmb.2012-0179OC

    Src inhibition prevents DHT from enhancing the activity of the EGFR. ( A – C ) PP2 inhibition of SRC prevents DHT from potentiating EGF signaling. Previously untreated primary fetal lung fibroblasts were exposed to PP2 before DHT treatment and EGF
    Figure Legend Snippet: Src inhibition prevents DHT from enhancing the activity of the EGFR. ( A – C ) PP2 inhibition of SRC prevents DHT from potentiating EGF signaling. Previously untreated primary fetal lung fibroblasts were exposed to PP2 before DHT treatment and EGF

    Techniques Used: Inhibition, Activity Assay

    DHT increases SRC phosphorylation and potentiates the ligand-dependent activation of the EGFR. EGF-induced SRC and EGFR pathway activation is potentiated by DHT. Previously untreated primary fetal lung fibroblasts were stimulated with 100 mg/ml EGF for
    Figure Legend Snippet: DHT increases SRC phosphorylation and potentiates the ligand-dependent activation of the EGFR. EGF-induced SRC and EGFR pathway activation is potentiated by DHT. Previously untreated primary fetal lung fibroblasts were stimulated with 100 mg/ml EGF for

    Techniques Used: Activation Assay

    8) Product Images from "The metastasis suppressor NDRG1 down-regulates the epidermal growth factor receptor via a lysosomal mechanism by up-regulating mitogen-inducible gene 6"

    Article Title: The metastasis suppressor NDRG1 down-regulates the epidermal growth factor receptor via a lysosomal mechanism by up-regulating mitogen-inducible gene 6

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.RA118.006279

    A and B , NDRG1 overexpression enhances MIG6 levels ( A ), with this effect being reversed ( B ) when NDRG1 is silenced. C, NDRG1's effect on the decreasing activated EGFR and up-regulating MIG6 is reversed in the presence of the lysosomotropic agents NH 4 Cl and CH 3 NH 2 . PANC-1 vector control (VC) and NDRG1-overexpressing (N1) cells were ( A ) incubated in the presence and absence of the ligand EGF (10 ng/ml) for 2, 5, and 10 min at 37 °C, and ( C ) with NH 4 Cl and CH 3 NH 2 , and in combination with EGF in the last 10 min of treatment at 37 °C. B , PANC-1, CFPAC-1, and AsPC-1 VC cells were transiently transfected with NDRG1 siRNA (siNDRG1) or nonspecific control siRNA (siControl) under the conditions described in “Materials and Methods.” Total cell protein was extracted and electrophoresed on a 10% SDS-PAGE gel followed by Western blot analysis to detect ( A ) and ( C ) NDRG1, EGFR, pEGFR (Tyr-1068), and MIG6 expression, and ( B ) NDRG1 and MIG6 expression. β-actin was used as a protein-loading control. Results are mean ± S.D. ( n = 3). A and C , *, p
    Figure Legend Snippet: A and B , NDRG1 overexpression enhances MIG6 levels ( A ), with this effect being reversed ( B ) when NDRG1 is silenced. C, NDRG1's effect on the decreasing activated EGFR and up-regulating MIG6 is reversed in the presence of the lysosomotropic agents NH 4 Cl and CH 3 NH 2 . PANC-1 vector control (VC) and NDRG1-overexpressing (N1) cells were ( A ) incubated in the presence and absence of the ligand EGF (10 ng/ml) for 2, 5, and 10 min at 37 °C, and ( C ) with NH 4 Cl and CH 3 NH 2 , and in combination with EGF in the last 10 min of treatment at 37 °C. B , PANC-1, CFPAC-1, and AsPC-1 VC cells were transiently transfected with NDRG1 siRNA (siNDRG1) or nonspecific control siRNA (siControl) under the conditions described in “Materials and Methods.” Total cell protein was extracted and electrophoresed on a 10% SDS-PAGE gel followed by Western blot analysis to detect ( A ) and ( C ) NDRG1, EGFR, pEGFR (Tyr-1068), and MIG6 expression, and ( B ) NDRG1 and MIG6 expression. β-actin was used as a protein-loading control. Results are mean ± S.D. ( n = 3). A and C , *, p

    Techniques Used: Over Expression, Plasmid Preparation, Incubation, Transfection, SDS Page, Western Blot, Expressing

    A–C , EGFR ( A and B ) does not directly associate with NDRG1, but ( C ) NDRG1 overexpression increases the half-life of MIG6. PANC-1 VC or N1 cells were incubated with control media or media containing EGF (10 ng/ml; 10 min/37 °C) and examined via co-immunoprecipitation. MIG6 ( A ) or NDRG1 ( B ) was immunoprecipitated and Western blotting performed to detect MIG6, EGFR, or NDRG1 expression. Input lysates of the total protein were included for comparison with immunoprecipitated lysates for each sample. Relative to untreated vector control cells: *, p
    Figure Legend Snippet: A–C , EGFR ( A and B ) does not directly associate with NDRG1, but ( C ) NDRG1 overexpression increases the half-life of MIG6. PANC-1 VC or N1 cells were incubated with control media or media containing EGF (10 ng/ml; 10 min/37 °C) and examined via co-immunoprecipitation. MIG6 ( A ) or NDRG1 ( B ) was immunoprecipitated and Western blotting performed to detect MIG6, EGFR, or NDRG1 expression. Input lysates of the total protein were included for comparison with immunoprecipitated lysates for each sample. Relative to untreated vector control cells: *, p

    Techniques Used: Over Expression, Incubation, Immunoprecipitation, Western Blot, Expressing, Plasmid Preparation

    Silencing of the tumor suppressors MIG6 and PTEN increases total and activated EGFR levels. A and B , PANC-1 VC or N1 cells were transiently transfected with nonspecific control siRNA (siControl) and ( A ) MIG6 siRNA (siMIG6) or ( B ) PTEN siRNA (siPTEN), followed by incubation with control media or this media containing EGF (10 ng/ml; 10 min/37 °C). Total cell protein was extracted and electrophoresed on a 10% SDS-PAGE gel followed by Western blot analysis to detect MIG6, NDRG1, EGFR, pEGFR (Tyr-1068), or PTEN expression. β-actin was used as a protein-loading control. Results are mean ± S.D. ( n = 3). *, p
    Figure Legend Snippet: Silencing of the tumor suppressors MIG6 and PTEN increases total and activated EGFR levels. A and B , PANC-1 VC or N1 cells were transiently transfected with nonspecific control siRNA (siControl) and ( A ) MIG6 siRNA (siMIG6) or ( B ) PTEN siRNA (siPTEN), followed by incubation with control media or this media containing EGF (10 ng/ml; 10 min/37 °C). Total cell protein was extracted and electrophoresed on a 10% SDS-PAGE gel followed by Western blot analysis to detect MIG6, NDRG1, EGFR, pEGFR (Tyr-1068), or PTEN expression. β-actin was used as a protein-loading control. Results are mean ± S.D. ( n = 3). *, p

    Techniques Used: Transfection, Incubation, SDS Page, Western Blot, Expressing

    A and B , NDRG1 increases EGFR internalization and co-localization with ( A ) the early endosome marker EEA1 in the presence of EGF and ( B ) the late endosome/lysosome marker LAMP2 in the absence and presence of EGF in PANC-1 cells. VC or N1 PANC-1 cells were incubated with either control medium or medium containing EGF (10 ng/ml) for 10 min/37 °C. Immunofluorescence images show staining for EGFR ( red ), DAPI for nuclei ( blue ) and ( A ) EEA1 ( green ) or ( B ) LAMP2 ( green ). All images were taken with a 63× objective and at the same exposure time using AxioVision™ software. Images are representative from three experiments performed. A and B , quantification of ( Ai ) pixel intensity of EGFR or EEA1, ( Aii ) co-localization of EGFR and EEA1, ( Bi ) pixel intensity of EGFR and LAMP2, and ( Bii ) co-localization of EGFR and LAMP2. The quantification was performed using ImageJ software and expressed as mean ± S.D. (three experiments) where *, p
    Figure Legend Snippet: A and B , NDRG1 increases EGFR internalization and co-localization with ( A ) the early endosome marker EEA1 in the presence of EGF and ( B ) the late endosome/lysosome marker LAMP2 in the absence and presence of EGF in PANC-1 cells. VC or N1 PANC-1 cells were incubated with either control medium or medium containing EGF (10 ng/ml) for 10 min/37 °C. Immunofluorescence images show staining for EGFR ( red ), DAPI for nuclei ( blue ) and ( A ) EEA1 ( green ) or ( B ) LAMP2 ( green ). All images were taken with a 63× objective and at the same exposure time using AxioVision™ software. Images are representative from three experiments performed. A and B , quantification of ( Ai ) pixel intensity of EGFR or EEA1, ( Aii ) co-localization of EGFR and EEA1, ( Bi ) pixel intensity of EGFR and LAMP2, and ( Bii ) co-localization of EGFR and LAMP2. The quantification was performed using ImageJ software and expressed as mean ± S.D. (three experiments) where *, p

    Techniques Used: Marker, Incubation, Immunofluorescence, Staining, Software

    Schematic illustrating the role of NDRG1 and MIG6 in facilitating EGFR processing by the lysosomal compartment. ). Notably, MIG6 binds to NDRG1 in the cytosol, with increased stabilization of MIG6 being observed in NDRG1 overexpressing cells. This increased half-life of MIG6 probably increases the access of MIG6 to EGFR to ensure down-regulation. However, NDRG1 does not bind to EGFR, with no association of MIG6 or NDRG1 being demonstrated with the late endosome/lysosomal marker, LAMP2. In contrast, NDRG1 expression increases the co-localization of EGFR with EEA1 and LAMP2. In summary, NDRG1 overexpression increases expression of the EGFR inhibitor MIG6 by stabilizing it, resulting in an MIG6-NDRG1 complex that potentiates EGFR down-regulation by enhanced lysosomal processing of EGFR.
    Figure Legend Snippet: Schematic illustrating the role of NDRG1 and MIG6 in facilitating EGFR processing by the lysosomal compartment. ). Notably, MIG6 binds to NDRG1 in the cytosol, with increased stabilization of MIG6 being observed in NDRG1 overexpressing cells. This increased half-life of MIG6 probably increases the access of MIG6 to EGFR to ensure down-regulation. However, NDRG1 does not bind to EGFR, with no association of MIG6 or NDRG1 being demonstrated with the late endosome/lysosomal marker, LAMP2. In contrast, NDRG1 expression increases the co-localization of EGFR with EEA1 and LAMP2. In summary, NDRG1 overexpression increases expression of the EGFR inhibitor MIG6 by stabilizing it, resulting in an MIG6-NDRG1 complex that potentiates EGFR down-regulation by enhanced lysosomal processing of EGFR.

    Techniques Used: Marker, Expressing, Over Expression

    9) Product Images from "Knockdown of HIP1 expression promotes ligand-induced endocytosis of EGFR in HeLa cells"

    Article Title: Knockdown of HIP1 expression promotes ligand-induced endocytosis of EGFR in HeLa cells

    Journal: Oncology Reports

    doi: 10.3892/or.2017.6025

    Endocytosis of EGFR and localization of clathrin in HeLa and HeLa HIP1 RNAi2 cells (100 ng/ml EGF stimulation). After 100 ng/ml EGF stimulation, endocytosis of EGF-bound EGFR and clathrin were accelerated significantly after the expression of HIP1 was blocked. EGFR and clathrin were colocalized in the cytoplasm.
    Figure Legend Snippet: Endocytosis of EGFR and localization of clathrin in HeLa and HeLa HIP1 RNAi2 cells (100 ng/ml EGF stimulation). After 100 ng/ml EGF stimulation, endocytosis of EGF-bound EGFR and clathrin were accelerated significantly after the expression of HIP1 was blocked. EGFR and clathrin were colocalized in the cytoplasm.

    Techniques Used: Expressing

    Endocytosis of EGFR and localization of clathrin in HeLa and HeLa HIP1 RNAi2 cells (1.5 ng/ml EGF stimulation). After 1.5 ng/ml EGF stimulation, endocytosis of EGF-bound EGFR and clathrin were accelerated after the expression of HIP1 was blocked. EGFR and clathrin were colocalized in the cytoplasm.
    Figure Legend Snippet: Endocytosis of EGFR and localization of clathrin in HeLa and HeLa HIP1 RNAi2 cells (1.5 ng/ml EGF stimulation). After 1.5 ng/ml EGF stimulation, endocytosis of EGF-bound EGFR and clathrin were accelerated after the expression of HIP1 was blocked. EGFR and clathrin were colocalized in the cytoplasm.

    Techniques Used: Expressing

    10) Product Images from "Visualization and quantitation of epidermal growth factor receptor homodimerization and activation with a proximity ligation assay"

    Article Title: Visualization and quantitation of epidermal growth factor receptor homodimerization and activation with a proximity ligation assay

    Journal: Oncotarget

    doi: 10.18632/oncotarget.19552

    Detection of EGFR homodimers in NSCLC cell lines by PLA analysis (A) PLA analysis of PC9 cells performed in the absence or presence of PLUS and MINUS probes as indicated. Red signals corresponding to EGFR homodimers were detected only in the presence of both probes. Nuclei were stained blue with 4′, 6-diamidino-2-phenylindole (DAPI). (B) PLA analysis of EGFR mutation–positive (PC9, HCC827) or –negative (A549) NSCLC cell lines.
    Figure Legend Snippet: Detection of EGFR homodimers in NSCLC cell lines by PLA analysis (A) PLA analysis of PC9 cells performed in the absence or presence of PLUS and MINUS probes as indicated. Red signals corresponding to EGFR homodimers were detected only in the presence of both probes. Nuclei were stained blue with 4′, 6-diamidino-2-phenylindole (DAPI). (B) PLA analysis of EGFR mutation–positive (PC9, HCC827) or –negative (A549) NSCLC cell lines.

    Techniques Used: Proximity Ligation Assay, Staining, Mutagenesis

    11) Product Images from "PGE2/EP4 receptor attenuated mucosal injury via β‐arrestin1/Src/EGFR‐mediated proliferation in portal hypertensive gastropathy) PGE2/EP4 receptor attenuated mucosal injury via β‐arrestin1/Src/EGFR‐mediated proliferation in portal hypertensive gastropathy"

    Article Title: PGE2/EP4 receptor attenuated mucosal injury via β‐arrestin1/Src/EGFR‐mediated proliferation in portal hypertensive gastropathy) PGE2/EP4 receptor attenuated mucosal injury via β‐arrestin1/Src/EGFR‐mediated proliferation in portal hypertensive gastropathy

    Journal: British Journal of Pharmacology

    doi: 10.1111/bph.13752

    Src and EGFR activations are suppressed in PHG. (A) Immunohistochemical staining for p‐EGFR and p‐Src from the representative uninvolved normal gastric mucosal tissue (from healthy volunteers) and gastropathic mucosal tissue from PHG patients is presented (brown, ×400, n = 6 per group). (B) Western blotting showed that p‐Src and p‐EGFR were repressed in PHG sections ( n = 6 per group). (C) and (D) The ratio of the normalized p‐Src/β‐actin and p‐EGFR/β‐actin was represented from western blotting ( n = 6 per group, Bonferroni's comparison post hoc test). (E) Immunofluorescence staining for p‐EGFR (red) with DAPI (blue) counterstaining for DNA in the indicated representative sections from PVL‐treated β‐arr1 ‐WT and β‐arr1 ‐KO mice, with or without PGE 2 treatment (3 mg·kg −1 ) (×400, n = 6 per group). (F) Immunofluorescence staining of p‐Src (red) was presented in the representative gastric mucosa of PVL‐treated mice; nuclei (blue) were counterstained with DAPI (×400, n = 6 per group).
    Figure Legend Snippet: Src and EGFR activations are suppressed in PHG. (A) Immunohistochemical staining for p‐EGFR and p‐Src from the representative uninvolved normal gastric mucosal tissue (from healthy volunteers) and gastropathic mucosal tissue from PHG patients is presented (brown, ×400, n = 6 per group). (B) Western blotting showed that p‐Src and p‐EGFR were repressed in PHG sections ( n = 6 per group). (C) and (D) The ratio of the normalized p‐Src/β‐actin and p‐EGFR/β‐actin was represented from western blotting ( n = 6 per group, Bonferroni's comparison post hoc test). (E) Immunofluorescence staining for p‐EGFR (red) with DAPI (blue) counterstaining for DNA in the indicated representative sections from PVL‐treated β‐arr1 ‐WT and β‐arr1 ‐KO mice, with or without PGE 2 treatment (3 mg·kg −1 ) (×400, n = 6 per group). (F) Immunofluorescence staining of p‐Src (red) was presented in the representative gastric mucosa of PVL‐treated mice; nuclei (blue) were counterstained with DAPI (×400, n = 6 per group).

    Techniques Used: Immunohistochemistry, Staining, Western Blot, Immunofluorescence, Mouse Assay

    PGE 2 /EP 4 receptor promotes Src and EGFR activation and complex formation via β‐arr1 in PHG. (A) Expression of the indicated proteins in the gastric mucosa of PVL‐treated β‐arr1 ‐WT and β‐arr1 ‐KO mice with or without PGE 2 treatment (3 mg·kg −1 ), as determined by western blotting. β‐actin was used as the loading control. n = 6 per group. (B) and (C) The ratio of densitometry units of the normalized p‐Src/β‐actin and p‐EGFR/β‐actin (EGFR) was represented from western blotting ( n = 6 per group, Bonferroni's comparison post hoc test). * P
    Figure Legend Snippet: PGE 2 /EP 4 receptor promotes Src and EGFR activation and complex formation via β‐arr1 in PHG. (A) Expression of the indicated proteins in the gastric mucosa of PVL‐treated β‐arr1 ‐WT and β‐arr1 ‐KO mice with or without PGE 2 treatment (3 mg·kg −1 ), as determined by western blotting. β‐actin was used as the loading control. n = 6 per group. (B) and (C) The ratio of densitometry units of the normalized p‐Src/β‐actin and p‐EGFR/β‐actin (EGFR) was represented from western blotting ( n = 6 per group, Bonferroni's comparison post hoc test). * P

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

    Inhibition of Src or EGFR blocks PGE 2 /EP 4 receptor‐mediated mucosal proliferation in PHG. (A) The Src inhibitor PP2 (80 nmol per mouse) significantly suppressed PVL‐induced p‐Src and p‐EGFR expression in mice, as determined by western blotting. n = 6 per group. (B) Western blotting revealed that the EGFR inhibitor AG1478 (80 nmol per mouse) suppressed PVL‐induced p‐EGFR expression in both PGE 2 ‐treated β‐arr1 ‐WT and β‐arr1 ‐KO mice under PVL. n = 6 per group. (C) and (D) PCNA‐stained images of the groups indicated (brown, ×200, n = 6 per group). (E) and (F) The proliferation index from PCNA staining was analysed as described previously. * P
    Figure Legend Snippet: Inhibition of Src or EGFR blocks PGE 2 /EP 4 receptor‐mediated mucosal proliferation in PHG. (A) The Src inhibitor PP2 (80 nmol per mouse) significantly suppressed PVL‐induced p‐Src and p‐EGFR expression in mice, as determined by western blotting. n = 6 per group. (B) Western blotting revealed that the EGFR inhibitor AG1478 (80 nmol per mouse) suppressed PVL‐induced p‐EGFR expression in both PGE 2 ‐treated β‐arr1 ‐WT and β‐arr1 ‐KO mice under PVL. n = 6 per group. (C) and (D) PCNA‐stained images of the groups indicated (brown, ×200, n = 6 per group). (E) and (F) The proliferation index from PCNA staining was analysed as described previously. * P

    Techniques Used: Inhibition, Expressing, Mouse Assay, Western Blot, Staining

    The PGE 2 /EP 4 receptor‐mediated β‐arr1/Src/EGFR complex contributes to mucosal proliferation via Akt activation in PHG. (A) PP2 treatment (80 nmol per mouse) blocked the upregulation of p‐Akt and PCNA in β‐arr1 ‐WT mice following PGE 2 administration (3 mg·kg −1 ) and promoted the expression of cleaved caspase‐3 in the representative mucosa of β‐arr1 ‐WT mice following PGE 2 administration. β‐actin was used as the loading control, n = 6 per group. (B) AG1478 treatment (80 nmol per mouse) down‐regulated the levels of p‐Akt and PCNA in β‐arr1 ‐WT mice following PGE 2 administration (3 mg·kg −1 ) and enhanced the expression of cleaved caspase‐3 in the mucosa of PVL‐treated β‐arr1 ‐WT mice with PGE 2 administration. β‐actin was used as the loading control, n = 6 per group. (C) and (D) The ratio of densitometry units of the normalized p‐Akt/β‐actin and PCNA/β‐actin of PP2‐treated sections from western blotting. * P
    Figure Legend Snippet: The PGE 2 /EP 4 receptor‐mediated β‐arr1/Src/EGFR complex contributes to mucosal proliferation via Akt activation in PHG. (A) PP2 treatment (80 nmol per mouse) blocked the upregulation of p‐Akt and PCNA in β‐arr1 ‐WT mice following PGE 2 administration (3 mg·kg −1 ) and promoted the expression of cleaved caspase‐3 in the representative mucosa of β‐arr1 ‐WT mice following PGE 2 administration. β‐actin was used as the loading control, n = 6 per group. (B) AG1478 treatment (80 nmol per mouse) down‐regulated the levels of p‐Akt and PCNA in β‐arr1 ‐WT mice following PGE 2 administration (3 mg·kg −1 ) and enhanced the expression of cleaved caspase‐3 in the mucosa of PVL‐treated β‐arr1 ‐WT mice with PGE 2 administration. β‐actin was used as the loading control, n = 6 per group. (C) and (D) The ratio of densitometry units of the normalized p‐Akt/β‐actin and PCNA/β‐actin of PP2‐treated sections from western blotting. * P

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

    Inhibition of Akt activation blocks β‐arr1/Src/EGFR‐mediated mucosal proliferation in PHG. (A) Treatment with the Akt inhibitor VIII (A6730, 50 mg·kg −1 ) down‐regulated p‐Akt and PCNA expression in β‐arr1 ‐WT mice following PGE 2 administration (3 mg·kg −1 ) but up‐regulated cleaved caspase‐3 expression in the mucosa of PVL‐treated β‐arr1 ‐WT mice administered PGE 2 . β‐actin was used as the loading control, n = 6 per group. (B) The Akt inhibitor VIII (50 mg·kg −1 ) did not affect the levels of p‐Src and p‐EGFR in PVL‐treated β‐arr1 ‐WT and β‐arr1 ‐KO mice following PGE 2 administration (3 mg·kg −1 ). β‐actin was used as the loading control, n = 6 per group. (C) and (D) The ratio of densitometry units of the normalized PCNA/β‐actin and cleaved caspase‐3/β‐actin of Akt inhibitor VIII‐treated sections from western blotting. * P
    Figure Legend Snippet: Inhibition of Akt activation blocks β‐arr1/Src/EGFR‐mediated mucosal proliferation in PHG. (A) Treatment with the Akt inhibitor VIII (A6730, 50 mg·kg −1 ) down‐regulated p‐Akt and PCNA expression in β‐arr1 ‐WT mice following PGE 2 administration (3 mg·kg −1 ) but up‐regulated cleaved caspase‐3 expression in the mucosa of PVL‐treated β‐arr1 ‐WT mice administered PGE 2 . β‐actin was used as the loading control, n = 6 per group. (B) The Akt inhibitor VIII (50 mg·kg −1 ) did not affect the levels of p‐Src and p‐EGFR in PVL‐treated β‐arr1 ‐WT and β‐arr1 ‐KO mice following PGE 2 administration (3 mg·kg −1 ). β‐actin was used as the loading control, n = 6 per group. (C) and (D) The ratio of densitometry units of the normalized PCNA/β‐actin and cleaved caspase‐3/β‐actin of Akt inhibitor VIII‐treated sections from western blotting. * P

    Techniques Used: Inhibition, Activation Assay, Expressing, Mouse Assay, Western Blot

    12) Product Images from "Zi Shen Huo Luo Formula Prevents Aldosterone-Induced Cardiomyocyte Hypertrophy and Cardiac Fibroblast Proliferation by Regulating the Striatin-Mediated MR/EGFR/ERK Signaling Pathway"

    Article Title: Zi Shen Huo Luo Formula Prevents Aldosterone-Induced Cardiomyocyte Hypertrophy and Cardiac Fibroblast Proliferation by Regulating the Striatin-Mediated MR/EGFR/ERK Signaling Pathway

    Journal: Evidence-based Complementary and Alternative Medicine : eCAM

    doi: 10.1155/2020/9028047

    Effects of ZSHLF on the protein expression levels of EGFR, ERK, pEGFR, and pERK in cardiomyocytes and cardiac fibroblasts. Expression levels of EGFR, ERK, and phosphorylated proteins in cardiomyocytes (a) and cardiac fibroblasts (b) were detected by Western blotting, and the ratio relative to β -actin expression was used as the relative target protein content (c). ▲ P
    Figure Legend Snippet: Effects of ZSHLF on the protein expression levels of EGFR, ERK, pEGFR, and pERK in cardiomyocytes and cardiac fibroblasts. Expression levels of EGFR, ERK, and phosphorylated proteins in cardiomyocytes (a) and cardiac fibroblasts (b) were detected by Western blotting, and the ratio relative to β -actin expression was used as the relative target protein content (c). ▲ P

    Techniques Used: Expressing, Western Blot

    13) Product Images from "NFATc4 Regulates Sox9 Gene Expression in Acinar Cell Plasticity and Pancreatic Cancer Initiation"

    Article Title: NFATc4 Regulates Sox9 Gene Expression in Acinar Cell Plasticity and Pancreatic Cancer Initiation

    Journal: Stem Cells International

    doi: 10.1155/2016/5272498

    NFATc4 is induced in acinar cell transdifferentiation. (a) Light microscopy shows morphology of acinar cell extracts from wildtype mice in response to TGF α for the indicated time points (scale bar represents 100 μ m). (b) qRT-PCR to detect mRNA expression of NFAT isoforms in transdifferentiating acinar cells of wildtype mice in response to TGF α . (c)-(d) NFATc4 mRNA (c) and protein (d) expression levels in Kras G12D ; p 53 Δ/ wt ; EGFR −/− ; pdx1-Cre mice were determined after transient overexpression of a control vector or constitutive EGFR, respectively. NFATc4 mRNA expression following EGFR overexpression was normalized to control (means ± SD). (e) qRT-PCR reveals mRNA expression of NFAT isoforms in primary tumor cells from Kras G12D ; p 53 Δ/ wt ; pdx1-Cre mice following TGF β treatment for 24 hours. (f) Western Blot analysis in primary tumor cells from Kras G12D ; p 53 Δ/ wt ; pdx1-Cre mice upon TGF β treatment. β -actin was utilized as a loading control. (g) Representative H E staining and immunohistochemistry were performed in human chronic pancreatitis samples. Scale bar represents 100 μ m. Asterisks indicate NFATc4 positive nuclei.
    Figure Legend Snippet: NFATc4 is induced in acinar cell transdifferentiation. (a) Light microscopy shows morphology of acinar cell extracts from wildtype mice in response to TGF α for the indicated time points (scale bar represents 100 μ m). (b) qRT-PCR to detect mRNA expression of NFAT isoforms in transdifferentiating acinar cells of wildtype mice in response to TGF α . (c)-(d) NFATc4 mRNA (c) and protein (d) expression levels in Kras G12D ; p 53 Δ/ wt ; EGFR −/− ; pdx1-Cre mice were determined after transient overexpression of a control vector or constitutive EGFR, respectively. NFATc4 mRNA expression following EGFR overexpression was normalized to control (means ± SD). (e) qRT-PCR reveals mRNA expression of NFAT isoforms in primary tumor cells from Kras G12D ; p 53 Δ/ wt ; pdx1-Cre mice following TGF β treatment for 24 hours. (f) Western Blot analysis in primary tumor cells from Kras G12D ; p 53 Δ/ wt ; pdx1-Cre mice upon TGF β treatment. β -actin was utilized as a loading control. (g) Representative H E staining and immunohistochemistry were performed in human chronic pancreatitis samples. Scale bar represents 100 μ m. Asterisks indicate NFATc4 positive nuclei.

    Techniques Used: Light Microscopy, Mouse Assay, Quantitative RT-PCR, Expressing, Over Expression, Plasmid Preparation, Western Blot, Staining, Immunohistochemistry

    14) Product Images from "HER2 induced EMT and tumorigenicity in breast epithelial progenitor cells is inhibited by coexpression of EGFR"

    Article Title: HER2 induced EMT and tumorigenicity in breast epithelial progenitor cells is inhibited by coexpression of EGFR

    Journal: Oncogene

    doi: 10.1038/onc.2015.489

    EGFR partially reverses HER2-induced EMT in 3D culture. ( a ) D492 HER2 cells show disrupted branching morphogenesis and generate spindle- and grape-like colonies in 3D culture. D492 cells expressing ctrl, HER2, EGFR and HER2/EGFR were seeded into rBM and cultured for 2 weeks. Top picture panel shows representative bright-field images of branching structures found in 3D organotypic cultures of D492 cells. Bar=100 μm. Representative isolated structures were fixed and stained for E- and N-cadherin, CK14 and CK19. Bar=50 μm. After the culture period, colonies were counted into four groups, branching/budding, solid round, grape like and spindle. The left table demonstrates the frequencies of each of these phenotypic groups within each D492 subline. The right table demonstrates the same ratios when cultures were treated with the small-molecule HER2 inhibitor CP724,714. ( b ) EGFR restores the epithelial phenotype in 3D culture in D492 HER2 (D492 HER2/EGFR ). Western immunoblot for E- and N-cadherin, CK14 and Ck19 of protein lysates isolated from 3D cultures after 14 days of cultivation.
    Figure Legend Snippet: EGFR partially reverses HER2-induced EMT in 3D culture. ( a ) D492 HER2 cells show disrupted branching morphogenesis and generate spindle- and grape-like colonies in 3D culture. D492 cells expressing ctrl, HER2, EGFR and HER2/EGFR were seeded into rBM and cultured for 2 weeks. Top picture panel shows representative bright-field images of branching structures found in 3D organotypic cultures of D492 cells. Bar=100 μm. Representative isolated structures were fixed and stained for E- and N-cadherin, CK14 and CK19. Bar=50 μm. After the culture period, colonies were counted into four groups, branching/budding, solid round, grape like and spindle. The left table demonstrates the frequencies of each of these phenotypic groups within each D492 subline. The right table demonstrates the same ratios when cultures were treated with the small-molecule HER2 inhibitor CP724,714. ( b ) EGFR restores the epithelial phenotype in 3D culture in D492 HER2 (D492 HER2/EGFR ). Western immunoblot for E- and N-cadherin, CK14 and Ck19 of protein lysates isolated from 3D cultures after 14 days of cultivation.

    Techniques Used: Expressing, Cell Culture, Isolation, Staining, Western Blot

    EGFR expression reverses HER2-induced loss of cytokeratin expression in xenografts tumors. ( a ) Mosaic picture of sections from H E staining of D492 HER2 and D492 HER2/EGFR tumors. Tumors were paraffin embedded, sliced and stained with H E. Squares denote areas represented in immunofluorescence in ( b ). Bar=1 mm. ( b ) EGFR reverses HER2-induced loss of cytokeratin expression. Confocal images showing xenograft slices from D492 HER2 and D492 HER2/EGFR tumors stained with HER2, EGFR, CK14 and E-cadherin. Bar=100 μm. ( c ) Apoptosis levels are higher in close proximity to epithelial, CK14-expressing tumor areas. Confocal images showing xenograft slices from D492 HER2 and D492 HER2/EGFR tumors stained with CK14, Ki67 and cleaved caspase-3 antibodies. Bar=100 μm.
    Figure Legend Snippet: EGFR expression reverses HER2-induced loss of cytokeratin expression in xenografts tumors. ( a ) Mosaic picture of sections from H E staining of D492 HER2 and D492 HER2/EGFR tumors. Tumors were paraffin embedded, sliced and stained with H E. Squares denote areas represented in immunofluorescence in ( b ). Bar=1 mm. ( b ) EGFR reverses HER2-induced loss of cytokeratin expression. Confocal images showing xenograft slices from D492 HER2 and D492 HER2/EGFR tumors stained with HER2, EGFR, CK14 and E-cadherin. Bar=100 μm. ( c ) Apoptosis levels are higher in close proximity to epithelial, CK14-expressing tumor areas. Confocal images showing xenograft slices from D492 HER2 and D492 HER2/EGFR tumors stained with CK14, Ki67 and cleaved caspase-3 antibodies. Bar=100 μm.

    Techniques Used: Expressing, Staining, Immunofluorescence

    HER2 overexpression induces mesenchymal transition. ( a ) D492 HER2 cells lose epithelial phenotype in monolayer culture. Confocal microscopy images of D492 cells expressing ctrl, HER2, EGFR and HER2/EGFR grown on culture flasks and analyzed by immunofluorescence staining for CK14, CK19, E-, N- and P-cadherin expression. Bar=50 μm. ( b ) D492 HER2 and D492 HER2/EGFR gain mesenchymal phenotype in monolayer culture. Western blotting performed using lysates from monolayer cultures. Membranes were blotted for CK14, CK19, p63, E-, P-, N-cadherin and Axl expression. Actin=Loading control. ( c ) In D492 HER2 , miR-200c and miR141 are strongly downregulated. Quantitative reverse transcriptase–PCR was performed using monolayer RNA isolates from all four cell lines. Transcription of miR-200c, miR-141 and ZEB1was analyzed and normalized to GAPDH.
    Figure Legend Snippet: HER2 overexpression induces mesenchymal transition. ( a ) D492 HER2 cells lose epithelial phenotype in monolayer culture. Confocal microscopy images of D492 cells expressing ctrl, HER2, EGFR and HER2/EGFR grown on culture flasks and analyzed by immunofluorescence staining for CK14, CK19, E-, N- and P-cadherin expression. Bar=50 μm. ( b ) D492 HER2 and D492 HER2/EGFR gain mesenchymal phenotype in monolayer culture. Western blotting performed using lysates from monolayer cultures. Membranes were blotted for CK14, CK19, p63, E-, P-, N-cadherin and Axl expression. Actin=Loading control. ( c ) In D492 HER2 , miR-200c and miR141 are strongly downregulated. Quantitative reverse transcriptase–PCR was performed using monolayer RNA isolates from all four cell lines. Transcription of miR-200c, miR-141 and ZEB1was analyzed and normalized to GAPDH.

    Techniques Used: Over Expression, Confocal Microscopy, Expressing, Immunofluorescence, Staining, Western Blot, Polymerase Chain Reaction

    15) Product Images from "Anti-SSTR2 peptide based targeted delivery of potent PLGA encapsulated 3,3’-diindolylmethane nanoparticles through blood brain barrier prevents glioma progression"

    Article Title: Anti-SSTR2 peptide based targeted delivery of potent PLGA encapsulated 3,3’-diindolylmethane nanoparticles through blood brain barrier prevents glioma progression

    Journal: Oncotarget

    doi: 10.18632/oncotarget.18689

    SSTR2pep-DIM-NP mediated inhibition of EGFR pathway in vivo Representative images of IHC analysis of (A) human and rat glioma samples (n=25 for each group) show EGFR and SSTR2 expression. (B) Scattered plot represents mean H-scores of EGFR and SSTR2 for both groups. Correlation coefficient (r) between mean H-scores are +0.2901 and +0.1504 for human and rat samples respectively. (C) IHC analysis for P-EGFR, P-AKT, P-ERK, P-STAT3, Bcl-XL, p21. (D) (E) Immunofluorescence study for cleaved caspase 3 (FITC-green) and cleaved PARP (TRITC-red). (F) TUNEL positive cells in untreated and SSTR2pep-DIM-NP treated rat brain tumors. All images were captured in an upright microscope with 10X magnification.
    Figure Legend Snippet: SSTR2pep-DIM-NP mediated inhibition of EGFR pathway in vivo Representative images of IHC analysis of (A) human and rat glioma samples (n=25 for each group) show EGFR and SSTR2 expression. (B) Scattered plot represents mean H-scores of EGFR and SSTR2 for both groups. Correlation coefficient (r) between mean H-scores are +0.2901 and +0.1504 for human and rat samples respectively. (C) IHC analysis for P-EGFR, P-AKT, P-ERK, P-STAT3, Bcl-XL, p21. (D) (E) Immunofluorescence study for cleaved caspase 3 (FITC-green) and cleaved PARP (TRITC-red). (F) TUNEL positive cells in untreated and SSTR2pep-DIM-NP treated rat brain tumors. All images were captured in an upright microscope with 10X magnification.

    Techniques Used: Inhibition, In Vivo, Immunohistochemistry, Expressing, Immunofluorescence, TUNEL Assay, Microscopy

    16) Product Images from "Epidermal growth factor receptor activity is elevated in glioma cancer stem cells and is required to maintain chemotherapy and radiation resistance"

    Article Title: Epidermal growth factor receptor activity is elevated in glioma cancer stem cells and is required to maintain chemotherapy and radiation resistance

    Journal: Oncotarget

    doi: 10.18632/oncotarget.19868

    EGFR is constitutively active in CSCs (A) J3T adherent and sphere cells were treated with 5 μM gefitinib and harvested over the indicated time course. Expression of EGFR, phosphorylation of EGFR at serine-1047 and tyrosine-1173, AKT, phosphorylation of AKT, and β-actin as a loading control. 20 μg was loaded per lane. Comparison of the effect of increasing doses of gefitinib on proliferation of adherent and spheres of J3T (B) and LN18 cells (C) . Cells were treated with increasing doses of gefitinib and cell viability was assayed 48 hours post treatment.
    Figure Legend Snippet: EGFR is constitutively active in CSCs (A) J3T adherent and sphere cells were treated with 5 μM gefitinib and harvested over the indicated time course. Expression of EGFR, phosphorylation of EGFR at serine-1047 and tyrosine-1173, AKT, phosphorylation of AKT, and β-actin as a loading control. 20 μg was loaded per lane. Comparison of the effect of increasing doses of gefitinib on proliferation of adherent and spheres of J3T (B) and LN18 cells (C) . Cells were treated with increasing doses of gefitinib and cell viability was assayed 48 hours post treatment.

    Techniques Used: Expressing

    17) Product Images from "Genomic and Molecular Characterization of Malignant Peripheral Nerve Sheath Tumor Identifies the IGF1R Pathway as a Primary Target for Treatment"

    Article Title: Genomic and Molecular Characterization of Malignant Peripheral Nerve Sheath Tumor Identifies the IGF1R Pathway as a Primary Target for Treatment

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

    doi: 10.1158/1078-0432.CCR-11-1707

    Attenuated IGF1R and/or EGFR significantly inhibited cell proliferation in MPNST ST88-14 cells by blocking the PI3K/AKT and MAPK pathways
    Figure Legend Snippet: Attenuated IGF1R and/or EGFR significantly inhibited cell proliferation in MPNST ST88-14 cells by blocking the PI3K/AKT and MAPK pathways

    Techniques Used: Blocking Assay

    18) 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

    Synergic effect of 4D3 anti-CLDN4 antibody with anti-EGFR antibody ( A ) Effect of 4D3 on growth inhibition induced by anti-EGFR antibody C225 in spheres of Caco-2 cells. Anti-mouse CLDN4 was treated as a negative control. ( B ) Permeation of Caco-2 cell spheres with antibodies. Spheres were treated with fluorescence-labeled 4D3 and C225. Nuclei were stained by DAPI. ( C ) Effect of co-treatment with 4D3 and C225 on phosphorylation of EGFR in Caco-2 cell spheres. ( D , E ) Effect of 4D3 on C225-induced inhibition of tumor growth in nude mice ( n = 5). ( F ) Effect of time interval (h) between treatment with 4D3 and treatment with C225 on phosphorylation of EGFR in spheres. ( G ) Effects of sequential (Seq, 6-h time interval) vs. synchronous (Syn) treatment with 4D3 and C225 on tumor growth of Caco-2 cells in nude mice ( n = 5).
    Figure Legend Snippet: Synergic effect of 4D3 anti-CLDN4 antibody with anti-EGFR antibody ( A ) Effect of 4D3 on growth inhibition induced by anti-EGFR antibody C225 in spheres of Caco-2 cells. Anti-mouse CLDN4 was treated as a negative control. ( B ) Permeation of Caco-2 cell spheres with antibodies. Spheres were treated with fluorescence-labeled 4D3 and C225. Nuclei were stained by DAPI. ( C ) Effect of co-treatment with 4D3 and C225 on phosphorylation of EGFR in Caco-2 cell spheres. ( D , E ) Effect of 4D3 on C225-induced inhibition of tumor growth in nude mice ( n = 5). ( F ) Effect of time interval (h) between treatment with 4D3 and treatment with C225 on phosphorylation of EGFR in spheres. ( G ) Effects of sequential (Seq, 6-h time interval) vs. synchronous (Syn) treatment with 4D3 and C225 on tumor growth of Caco-2 cells in nude mice ( n = 5).

    Techniques Used: Inhibition, Negative Control, Fluorescence, Labeling, Staining, Mouse Assay

    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

    19) 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

    20) Product Images from "Epstein-Barr virus-encoded LMP2A stimulates migration of nasopharyngeal carcinoma cells via the EGFR/Ca2+/calpain/ITGβ4 axis"

    Article Title: Epstein-Barr virus-encoded LMP2A stimulates migration of nasopharyngeal carcinoma cells via the EGFR/Ca2+/calpain/ITGβ4 axis

    Journal: Biology Open

    doi: 10.1242/bio.024646

    Calcium chelator inhibits calpain activity and ITGβ4 cleavage in LMP2A-expressing NPC cells. (A) Calpain activity measured in a fluorometric assay in LMP2A-positive CNE1/TW03 cell lines before and after treatment with BAPTA-AM (10 μm/l) for 24 h. Data are mean±s.d. ( n =3). (B) Top panel: immunoblots showing the expression of ITGβ4 (205 kDa), cleaved ITGβ4 isoforms (165/125 kDa) and tyrosine-phosphorylated EGFR in LMP2A-expressing and control cells. GAPDH was used as loading control. Bottom panel: semi-quantitative analysis of three repeated immunoblot experiments as in the top panel. Relative expression of the cleaved ITGβ4 isoforms (left) and pEGFR (right) under the same experimental conditions. ** P
    Figure Legend Snippet: Calcium chelator inhibits calpain activity and ITGβ4 cleavage in LMP2A-expressing NPC cells. (A) Calpain activity measured in a fluorometric assay in LMP2A-positive CNE1/TW03 cell lines before and after treatment with BAPTA-AM (10 μm/l) for 24 h. Data are mean±s.d. ( n =3). (B) Top panel: immunoblots showing the expression of ITGβ4 (205 kDa), cleaved ITGβ4 isoforms (165/125 kDa) and tyrosine-phosphorylated EGFR in LMP2A-expressing and control cells. GAPDH was used as loading control. Bottom panel: semi-quantitative analysis of three repeated immunoblot experiments as in the top panel. Relative expression of the cleaved ITGβ4 isoforms (left) and pEGFR (right) under the same experimental conditions. ** P

    Techniques Used: Activity Assay, Expressing, Western Blot

    21) Product Images from "Immobilized epidermal growth factor stimulates persistent, directed keratinocyte migration via activation of PLCγ1"

    Article Title: Immobilized epidermal growth factor stimulates persistent, directed keratinocyte migration via activation of PLCγ1

    Journal: The FASEB Journal

    doi: 10.1096/fj.201600252

    Changes in EGFR localization in response to soluble or immobilized EGF. Immunofluorescent staining for EGFR (green) and EEA1 (red); blue is nuclear stain. Red line indicates the cellular plane shown in the panels. Immob, immobilized; sol, soluble. Scale
    Figure Legend Snippet: Changes in EGFR localization in response to soluble or immobilized EGF. Immunofluorescent staining for EGFR (green) and EEA1 (red); blue is nuclear stain. Red line indicates the cellular plane shown in the panels. Immob, immobilized; sol, soluble. Scale

    Techniques Used: Staining

    22) Product Images from "CUL4A overexpression enhances lung tumor growth and sensitizes lung cancer cells to Erlotinib via transcriptional regulation of EGFR"

    Article Title: CUL4A overexpression enhances lung tumor growth and sensitizes lung cancer cells to Erlotinib via transcriptional regulation of EGFR

    Journal: Molecular Cancer

    doi: 10.1186/1476-4598-13-252

    CUL4A regulates EGFR expression. (A) RT-PCR analysis of the expression of EGFR mRNA in H1299, H1650, A549 and H460 cells. (B) Western blot analysis of the expression of EGFR protein in H1299, H1650, A549 and H460 cells. (C) Immunofluorescence microscopy analysis of EGFR expression of in H1299, H1650, A549 and H460 cells. (D) The immunohistochemistry analysis of CUL4A and EGFR expression in NSCLC biopsy showed that CUL4A levels significantly correlate with EGFR levels in NSCLC tissues. All results are from three independent experiments. Scale bar indicates 20 μm (C) , and 50 μm (D) .
    Figure Legend Snippet: CUL4A regulates EGFR expression. (A) RT-PCR analysis of the expression of EGFR mRNA in H1299, H1650, A549 and H460 cells. (B) Western blot analysis of the expression of EGFR protein in H1299, H1650, A549 and H460 cells. (C) Immunofluorescence microscopy analysis of EGFR expression of in H1299, H1650, A549 and H460 cells. (D) The immunohistochemistry analysis of CUL4A and EGFR expression in NSCLC biopsy showed that CUL4A levels significantly correlate with EGFR levels in NSCLC tissues. All results are from three independent experiments. Scale bar indicates 20 μm (C) , and 50 μm (D) .

    Techniques Used: Expressing, Reverse Transcription Polymerase Chain Reaction, Western Blot, Immunofluorescence, Microscopy, Immunohistochemistry

    CUL4A transcriptionally activates EGFR expression in NSCLC tissues. (A) Western blot analysis of H3K4me3 levels in H1299-pBabe, H1299-CUL4A, A549-pSuper, and A549-shCUL4A cells. (B) Schematic presentation of two regions relative to the EGFR transcriptional start site used as primers to test H3K4me3 occupied abundance. (C) ChIP-PCR was performed to assess H3K4me3 occupancy in EGFR promoter in H1299-pBabe and H1299-CUL4A cells. (D) ChIP-PCR was performed to assess H3K4me3 occupancy in EGFR promoter in A549-pSuper and A549-shCUL4A cells. IgG was used as negative control.
    Figure Legend Snippet: CUL4A transcriptionally activates EGFR expression in NSCLC tissues. (A) Western blot analysis of H3K4me3 levels in H1299-pBabe, H1299-CUL4A, A549-pSuper, and A549-shCUL4A cells. (B) Schematic presentation of two regions relative to the EGFR transcriptional start site used as primers to test H3K4me3 occupied abundance. (C) ChIP-PCR was performed to assess H3K4me3 occupancy in EGFR promoter in H1299-pBabe and H1299-CUL4A cells. (D) ChIP-PCR was performed to assess H3K4me3 occupancy in EGFR promoter in A549-pSuper and A549-shCUL4A cells. IgG was used as negative control.

    Techniques Used: Expressing, Western Blot, Chromatin Immunoprecipitation, Polymerase Chain Reaction, Negative Control

    CUL4A activates the EGFR-mediated signaling pathways. (A) Levels of CUL4A, EGFR, p-EGFR, p-AKT, and AKT were analyzed by Western blot in H1299-pBabe, H1299-CUL4A, H1650-pBabe and H1650-CUL4A cells. (B) Levels of CUL4A, EGFR, p-EGFR, p-AKT, and AKT were analyzed by Western blot in A549-pSuper, A549-shCUL4A, H460-pSuper and H460-shCUL4A cells. (C) Western blot to analyze the effect of erlotinib on the levels of CUL4A, EGFR, p-EGFR, p-AKT, and AKT in H1299-pBabe and H1299-CUL4A cells. (D) MTT analysis of the inhibition of erlotinib on cell proliferation in CUL4A overexprssion cells (H1299-CUL4A and H1650-CUL4A). ** P
    Figure Legend Snippet: CUL4A activates the EGFR-mediated signaling pathways. (A) Levels of CUL4A, EGFR, p-EGFR, p-AKT, and AKT were analyzed by Western blot in H1299-pBabe, H1299-CUL4A, H1650-pBabe and H1650-CUL4A cells. (B) Levels of CUL4A, EGFR, p-EGFR, p-AKT, and AKT were analyzed by Western blot in A549-pSuper, A549-shCUL4A, H460-pSuper and H460-shCUL4A cells. (C) Western blot to analyze the effect of erlotinib on the levels of CUL4A, EGFR, p-EGFR, p-AKT, and AKT in H1299-pBabe and H1299-CUL4A cells. (D) MTT analysis of the inhibition of erlotinib on cell proliferation in CUL4A overexprssion cells (H1299-CUL4A and H1650-CUL4A). ** P

    Techniques Used: Western Blot, MTT Assay, Inhibition

    23) Product Images from "Role of HER3 expression and PTEN loss in patients with HER2-overexpressing metastatic breast cancer (MBC) who received taxane plus trastuzumab treatment"

    Article Title: Role of HER3 expression and PTEN loss in patients with HER2-overexpressing metastatic breast cancer (MBC) who received taxane plus trastuzumab treatment

    Journal: British Journal of Cancer

    doi: 10.1038/bjc.2013.757

    Kaplan–Meier OS curves according to IHC. ( A ) OS according to ER status. ( B ) OS according to HER3 status. ( C ) OS according to PTEN status. ( D ) OS according to EGFR status.
    Figure Legend Snippet: Kaplan–Meier OS curves according to IHC. ( A ) OS according to ER status. ( B ) OS according to HER3 status. ( C ) OS according to PTEN status. ( D ) OS according to EGFR status.

    Techniques Used: Immunohistochemistry

    Kaplan–Meier PFS curves according to IHC. ( A ) PFS according to ER status. ( B ) PFS according to HER3 status. ( C ) PFS according to PTEN status. ( D ) PFS according to EGFR status.
    Figure Legend Snippet: Kaplan–Meier PFS curves according to IHC. ( A ) PFS according to ER status. ( B ) PFS according to HER3 status. ( C ) PFS according to PTEN status. ( D ) PFS according to EGFR status.

    Techniques Used: Immunohistochemistry

    24) Product Images from "Radiotherapy Upregulates Programmed Death Ligand-1 through the Pathways Downstream of Epidermal Growth Factor Receptor in Glioma"

    Article Title: Radiotherapy Upregulates Programmed Death Ligand-1 through the Pathways Downstream of Epidermal Growth Factor Receptor in Glioma

    Journal: EBioMedicine

    doi: 10.1016/j.ebiom.2018.01.027

    EGFR inhibitor abrogates the up-regulation of PD-L1 at the mRNA level caused by RT in glioma cells. Cell lines (U87 and U251) were treated with vehicle control, RT (5 or 10 Gy), or RT combined with AG490 (10 μM) for 24, 48 or 72 h. PD-L1 expression at the mRNA level was determined by RT-PCR and expressed as fold change relative to the vehicle group. Each column is shown as the means of three separate experiments; bars, SD. ANOVA; *** P
    Figure Legend Snippet: EGFR inhibitor abrogates the up-regulation of PD-L1 at the mRNA level caused by RT in glioma cells. Cell lines (U87 and U251) were treated with vehicle control, RT (5 or 10 Gy), or RT combined with AG490 (10 μM) for 24, 48 or 72 h. PD-L1 expression at the mRNA level was determined by RT-PCR and expressed as fold change relative to the vehicle group. Each column is shown as the means of three separate experiments; bars, SD. ANOVA; *** P

    Techniques Used: Expressing, Reverse Transcription Polymerase Chain Reaction

    EGFR inhibitor abrogates the up-regulation of PD-L1 caused by RT in glioma cells. EGFR signaling and PD-L1 protein level in U87 and U251 cell lines were detected at 24, 48 and 72 h after 5 or 10 Gy irradiation. Before irradiation, U87 and U251 cells were incubated in the presence of 10 μM AG490 at 37 °C for 1 h. Each column is shown as the means of three separate experiments; bars, SD. ANOVA; * P
    Figure Legend Snippet: EGFR inhibitor abrogates the up-regulation of PD-L1 caused by RT in glioma cells. EGFR signaling and PD-L1 protein level in U87 and U251 cell lines were detected at 24, 48 and 72 h after 5 or 10 Gy irradiation. Before irradiation, U87 and U251 cells were incubated in the presence of 10 μM AG490 at 37 °C for 1 h. Each column is shown as the means of three separate experiments; bars, SD. ANOVA; * P

    Techniques Used: Irradiation, Incubation

    25) Product Images from "Increased Expression of Epidermal Growth Factor Receptor (EGF-R) in Patients with Different Forms of Lung Fibrosis"

    Article Title: Increased Expression of Epidermal Growth Factor Receptor (EGF-R) in Patients with Different Forms of Lung Fibrosis

    Journal: BioMed Research International

    doi: 10.1155/2013/654354

    Representative tissue microarray section immunostained with monoclonal antibody EGFR demonstrating diffuse cytoplasmic reaction of weak intensity (light brown) within alveolar epithelium (arrows) immediately surrounding areas of inflammation in patients with cellular NSIP ( n = 4), evidence that was further verified by double immunohistochemistry analysis revealing weak colocalization (light brown) of EGFR and SP-A (arrows).
    Figure Legend Snippet: Representative tissue microarray section immunostained with monoclonal antibody EGFR demonstrating diffuse cytoplasmic reaction of weak intensity (light brown) within alveolar epithelium (arrows) immediately surrounding areas of inflammation in patients with cellular NSIP ( n = 4), evidence that was further verified by double immunohistochemistry analysis revealing weak colocalization (light brown) of EGFR and SP-A (arrows).

    Techniques Used: Microarray, Immunohistochemistry

    (a) Computerized image analysis verified results from immunohistochemistry analysis demonstrating a statistically significant increased expression of EGFR in patients with fibrotic forms of IIPs compared to the inflammatory component of cellular NSIP as well as control lung samples. * P
    Figure Legend Snippet: (a) Computerized image analysis verified results from immunohistochemistry analysis demonstrating a statistically significant increased expression of EGFR in patients with fibrotic forms of IIPs compared to the inflammatory component of cellular NSIP as well as control lung samples. * P

    Techniques Used: Immunohistochemistry, Expressing

    Representative tissue microarray section immunostained with monoclonal antibody EGFR demonstrating diffuse cytoplasmic reaction (dark brown) of strong intensity within hyperplastic alveolar epithelium (arrows) immediately adjacent to Masson bodies in patients with COP ( n = 15), evidence that was further verified by double immunohistochemistry analysis revealing strong colocalization (light brown with red) of EGFR and SP-A (arrows).
    Figure Legend Snippet: Representative tissue microarray section immunostained with monoclonal antibody EGFR demonstrating diffuse cytoplasmic reaction (dark brown) of strong intensity within hyperplastic alveolar epithelium (arrows) immediately adjacent to Masson bodies in patients with COP ( n = 15), evidence that was further verified by double immunohistochemistry analysis revealing strong colocalization (light brown with red) of EGFR and SP-A (arrows).

    Techniques Used: Microarray, Immunohistochemistry

    Representative tissue microarray section immunostained with monoclonal antibody EGFR demonstrating diffuse cytoplasmic reaction (dark brown) of strong intensity within hyperplastic alveolar epithelium (arrows) immediately adjacent to fibrotic areas in patients with fibrotic NSIP ( n = 11), evidence that was further verified by double immunohistochemistry analysis revealing strong colocalization (light brown with red) of EGFR and SP-A (arrows).
    Figure Legend Snippet: Representative tissue microarray section immunostained with monoclonal antibody EGFR demonstrating diffuse cytoplasmic reaction (dark brown) of strong intensity within hyperplastic alveolar epithelium (arrows) immediately adjacent to fibrotic areas in patients with fibrotic NSIP ( n = 11), evidence that was further verified by double immunohistochemistry analysis revealing strong colocalization (light brown with red) of EGFR and SP-A (arrows).

    Techniques Used: Microarray, Immunohistochemistry

    Representative tissue microarray section immunostained with monoclonal antibody EGFR demonstrating diffuse cytoplasmic reaction (dark brown) of strong intensity within hyperplastic alveolar epithelium (arrows) immediately adjacent to fibroblastic foci in patients with IPF ( n = 20), evidence that was further verified by double immunohistochemistry analysis revealing strong colocalization (light brown with red) of EGFR and SP-A (arrows).
    Figure Legend Snippet: Representative tissue microarray section immunostained with monoclonal antibody EGFR demonstrating diffuse cytoplasmic reaction (dark brown) of strong intensity within hyperplastic alveolar epithelium (arrows) immediately adjacent to fibroblastic foci in patients with IPF ( n = 20), evidence that was further verified by double immunohistochemistry analysis revealing strong colocalization (light brown with red) of EGFR and SP-A (arrows).

    Techniques Used: Microarray, Immunohistochemistry

    26) Product Images from "MicroRNA 199a-5p Attenuates Retrograde Transport and Protects against Toxin-Induced Inhibition of Protein Biosynthesis"

    Article Title: MicroRNA 199a-5p Attenuates Retrograde Transport and Protects against Toxin-Induced Inhibition of Protein Biosynthesis

    Journal: Molecular and Cellular Biology

    doi: 10.1128/MCB.00548-17

    miR-199a-5p regulates endolysosomal trafficking. (A) Confocal microscopy immunofluorescence images showing subcellular localization of CD63 subjected to LysoTracker internalization for 30 min at 37°C in HeLa cells transfected with CM or miR-199a-5p. Magnif, magnification of areas in dashed boxes. Scale bar = 15 μm. (B) Flow cytometry analysis of LysoTracker internalization at 37°C in HeLa cells treated as described in the legend to panel A. Data are expressed as the geometric mean values (Gmean) from three independent experiments. Data are expressed as geometric mean values (Gmean) ± SEM from three independent experiments. (C) Results of flow cytometry analysis of CM- or miR-199a-5p-transfected HeLa cells that were cultured in the presence of 200 μg/ml fluorescent DQ green BSA for 1 h at 37°C and then incubated at 37°C for 2 h. Data are expressed as fold change of Gmean ± SEM. (D) HeLa cells were transfected with CM and miR-199a-5p, and 24 h after transfection, cells were rinsed with PBS and incubated in serum-free culture medium for 24 h. The medium was collected and precipitated with trichloroacetic acid (TCA), and the resulting pellets were analyzed by 4-to-20% acrylamide gradient SDS-PAGE and immunoblotted with rabbit polyclonal antibody against cathepsin D (CatD). Blots were also probed with antibody to Hsp90 as a loading control. The positions of molecular weight markers (MW; in thousands) and of the precursor (p), intermediate (i), and mature (m) forms of cathepsin D are indicated. Bottom, quantification of the results of 3 independent experiments. (E) Western blot analysis of EGFR, p62, LC3B-I/LC3B-II, and Vps26A protein levels in CM- and miR-199a-5p-transfected cells. Bottom, quantification of the results. (F) EGFR degradation assay in HeLa cells transfected with CM or miR-199a-5p and stimulated with EGF for the indicated times. Right, quantification of the results. (D to F) Error bars indicate SEM. *, P ≤ 0.05. (G) Immunofluorescence analysis of LC3B in HeLa cells transfected with CM or miR-199a-5p. Scale bar = 15 μm.
    Figure Legend Snippet: miR-199a-5p regulates endolysosomal trafficking. (A) Confocal microscopy immunofluorescence images showing subcellular localization of CD63 subjected to LysoTracker internalization for 30 min at 37°C in HeLa cells transfected with CM or miR-199a-5p. Magnif, magnification of areas in dashed boxes. Scale bar = 15 μm. (B) Flow cytometry analysis of LysoTracker internalization at 37°C in HeLa cells treated as described in the legend to panel A. Data are expressed as the geometric mean values (Gmean) from three independent experiments. Data are expressed as geometric mean values (Gmean) ± SEM from three independent experiments. (C) Results of flow cytometry analysis of CM- or miR-199a-5p-transfected HeLa cells that were cultured in the presence of 200 μg/ml fluorescent DQ green BSA for 1 h at 37°C and then incubated at 37°C for 2 h. Data are expressed as fold change of Gmean ± SEM. (D) HeLa cells were transfected with CM and miR-199a-5p, and 24 h after transfection, cells were rinsed with PBS and incubated in serum-free culture medium for 24 h. The medium was collected and precipitated with trichloroacetic acid (TCA), and the resulting pellets were analyzed by 4-to-20% acrylamide gradient SDS-PAGE and immunoblotted with rabbit polyclonal antibody against cathepsin D (CatD). Blots were also probed with antibody to Hsp90 as a loading control. The positions of molecular weight markers (MW; in thousands) and of the precursor (p), intermediate (i), and mature (m) forms of cathepsin D are indicated. Bottom, quantification of the results of 3 independent experiments. (E) Western blot analysis of EGFR, p62, LC3B-I/LC3B-II, and Vps26A protein levels in CM- and miR-199a-5p-transfected cells. Bottom, quantification of the results. (F) EGFR degradation assay in HeLa cells transfected with CM or miR-199a-5p and stimulated with EGF for the indicated times. Right, quantification of the results. (D to F) Error bars indicate SEM. *, P ≤ 0.05. (G) Immunofluorescence analysis of LC3B in HeLa cells transfected with CM or miR-199a-5p. Scale bar = 15 μm.

    Techniques Used: Confocal Microscopy, Immunofluorescence, Transfection, Flow Cytometry, Cytometry, Cell Culture, Incubation, SDS Page, Molecular Weight, Western Blot, Degradation Assay

    27) Product Images from "SSRP1 silencing inhibits the proliferation and malignancy of human glioma cells via the MAPK signaling pathway"

    Article Title: SSRP1 silencing inhibits the proliferation and malignancy of human glioma cells via the MAPK signaling pathway

    Journal: Oncology Reports

    doi: 10.3892/or.2017.5982

    Structure-specific recognition protein 1 (SSRP1) regulates the expression of proliferation- and migration-associated genes in glioma via the MAPK pathway. (A and B) Knockdown of SSRP1 expression decreased the expression of proliferation-associated genes, including p65, c-myc, cyclin D and E. The expression of EGFR, VEGF and Snail, proteins involved in migration, were also suppressed. (C and D) The downregulated expression of SSRP1 significantly decreased the total and phosphorylated protein levels of p38 and ERK, and only the phosphorylated protein levels of JNK.
    Figure Legend Snippet: Structure-specific recognition protein 1 (SSRP1) regulates the expression of proliferation- and migration-associated genes in glioma via the MAPK pathway. (A and B) Knockdown of SSRP1 expression decreased the expression of proliferation-associated genes, including p65, c-myc, cyclin D and E. The expression of EGFR, VEGF and Snail, proteins involved in migration, were also suppressed. (C and D) The downregulated expression of SSRP1 significantly decreased the total and phosphorylated protein levels of p38 and ERK, and only the phosphorylated protein levels of JNK.

    Techniques Used: Expressing, Migration

    28) Product Images from "MicroRNA-153 Inhibits Osteosarcoma Cells Proliferation and Invasion by Targeting TGF-β2"

    Article Title: MicroRNA-153 Inhibits Osteosarcoma Cells Proliferation and Invasion by Targeting TGF-β2

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0119225

    MiR-153 Overexpression repressed the p-SMAD2, p-SMAD3, EGFR and IGFBP-3 expression. The MG-63 was treated in serum-free medium in the presence and absence of TGF-β (50 ng/ml), scramble, or miRNA-153 mimic for 24 h. (A) Expression of p-SMAD2, t-SMAD2, p-SMAD3 and t-SMAD3 was detected using western blotting. GAPDH was used as a loading control. (B) Expression of EGFR and IGFBP-3 was detected using western blotting. GAPDH was used as a loading control. (C) The mRNA expression of EGFR was detected using qRT-PCR. The expression of MMP9 was normalized to GAPDH. (D) The mRNA Expression of IGFBP-3 was detected using qRT-PCR. The expression of EGFR was normalized to GAPDH.
    Figure Legend Snippet: MiR-153 Overexpression repressed the p-SMAD2, p-SMAD3, EGFR and IGFBP-3 expression. The MG-63 was treated in serum-free medium in the presence and absence of TGF-β (50 ng/ml), scramble, or miRNA-153 mimic for 24 h. (A) Expression of p-SMAD2, t-SMAD2, p-SMAD3 and t-SMAD3 was detected using western blotting. GAPDH was used as a loading control. (B) Expression of EGFR and IGFBP-3 was detected using western blotting. GAPDH was used as a loading control. (C) The mRNA expression of EGFR was detected using qRT-PCR. The expression of MMP9 was normalized to GAPDH. (D) The mRNA Expression of IGFBP-3 was detected using qRT-PCR. The expression of EGFR was normalized to GAPDH.

    Techniques Used: Over Expression, Expressing, Western Blot, Quantitative RT-PCR

    29) Product Images from "BRCA1/p220 loss triggers BRCA1-IRIS overexpression via mRNA stabilization in breast cancer cells"

    Article Title: BRCA1/p220 loss triggers BRCA1-IRIS overexpression via mRNA stabilization in breast cancer cells

    Journal: Oncotarget

    doi:

    The effect of BRCA1-IRIS and BRCA1/p220 on the expression of several TN/BL markers Expression of the indicated TN/BL markers mRNAs (left) or proteins (right) in BRCA1/p220-silenced or BRCA1/p220 and BRCA1-IRIS co-silenced HME cells (A), BRCA1-IRIS-silenced SUM149 cells (B), BRCA1/p220 overexpressing or BRCA1-IRIS silenced HCC1937 cells (C). (D) PCR analysis showing promoters (left) or 10kb upstream regions (right) of CK5, CK17, CDH3, EGFR, cyclin E, AUF1 and pCBP2 in BRCA1-IRIS or BRCA1/p220 immunoprecipitation from cross-linked HME cells using mono-specific antibodies. (E) ChIP analysis of the promoters of the indicated TN/BL markers in control- or c-Jun-silenced MDAMB231 cells or control- or Oct1-silenced HME cells. (F) Analysis of the indicated promoters activation in HME cells (left) or MDAMB231 (right) depleted from BRCA1/p220, Oct1, BRCA1-IRIS or c-Jun. Inset is the effect of each siRNA on its cognate protein in HME cells.
    Figure Legend Snippet: The effect of BRCA1-IRIS and BRCA1/p220 on the expression of several TN/BL markers Expression of the indicated TN/BL markers mRNAs (left) or proteins (right) in BRCA1/p220-silenced or BRCA1/p220 and BRCA1-IRIS co-silenced HME cells (A), BRCA1-IRIS-silenced SUM149 cells (B), BRCA1/p220 overexpressing or BRCA1-IRIS silenced HCC1937 cells (C). (D) PCR analysis showing promoters (left) or 10kb upstream regions (right) of CK5, CK17, CDH3, EGFR, cyclin E, AUF1 and pCBP2 in BRCA1-IRIS or BRCA1/p220 immunoprecipitation from cross-linked HME cells using mono-specific antibodies. (E) ChIP analysis of the promoters of the indicated TN/BL markers in control- or c-Jun-silenced MDAMB231 cells or control- or Oct1-silenced HME cells. (F) Analysis of the indicated promoters activation in HME cells (left) or MDAMB231 (right) depleted from BRCA1/p220, Oct1, BRCA1-IRIS or c-Jun. Inset is the effect of each siRNA on its cognate protein in HME cells.

    Techniques Used: Expressing, Polymerase Chain Reaction, Immunoprecipitation, Chromatin Immunoprecipitation, Activation Assay

    30) Product Images from "Differential Expression of RBM5, EGFR and KRAS mRNA and protein in non-small cell lung cancer tissues"

    Article Title: Differential Expression of RBM5, EGFR and KRAS mRNA and protein in non-small cell lung cancer tissues

    Journal: Journal of Experimental & Clinical Cancer Research : CR

    doi: 10.1186/1756-9966-31-36

    Expression of RBM5, EGFR and KRAS protein in NSCLC . A, Western blot of RBM5, EGFR and KRAS protein expression in representative tissue samples from NSCLC and non-tumor specimens. Total cellular protein was extracted, subjected to Western blot analysis and quantified using Quantity One software. B, Quantitative data from A. *p
    Figure Legend Snippet: Expression of RBM5, EGFR and KRAS protein in NSCLC . A, Western blot of RBM5, EGFR and KRAS protein expression in representative tissue samples from NSCLC and non-tumor specimens. Total cellular protein was extracted, subjected to Western blot analysis and quantified using Quantity One software. B, Quantitative data from A. *p

    Techniques Used: Expressing, Western Blot, Software

    Expression of RBM5, EGFR and KRAS mRNA in NSCLC . A, Agarose gel of semi-quantitative RT-PCR data of RBM5, EGFR, and KRAS mRNA expression in representative NSCLC and non-tumor specimens. Total RNA was isolated and subjected to semi-quantitative RT-PCR and quantified using Quantity One software. B , Quantitative data from A. *p
    Figure Legend Snippet: Expression of RBM5, EGFR and KRAS mRNA in NSCLC . A, Agarose gel of semi-quantitative RT-PCR data of RBM5, EGFR, and KRAS mRNA expression in representative NSCLC and non-tumor specimens. Total RNA was isolated and subjected to semi-quantitative RT-PCR and quantified using Quantity One software. B , Quantitative data from A. *p

    Techniques Used: Expressing, Agarose Gel Electrophoresis, Quantitative RT-PCR, Isolation, Software

    31) Product Images from "Protective Effect of Ocotillol, the Derivate of Ocotillol-Type Saponins in Panax Genus, against Acetic Acid-Induced Gastric Ulcer in Rats Based on Untargeted Metabolomics"

    Article Title: Protective Effect of Ocotillol, the Derivate of Ocotillol-Type Saponins in Panax Genus, against Acetic Acid-Induced Gastric Ulcer in Rats Based on Untargeted Metabolomics

    Journal: International Journal of Molecular Sciences

    doi: 10.3390/ijms21072577

    ( A ) Immunohistochemical analysis of ET-1, NOS2 and EGFR in different groups (×100). OD values of ( B ) ET-1, ( C ) NOS2 and ( D ) EGFR (Compared with normal group, ## p
    Figure Legend Snippet: ( A ) Immunohistochemical analysis of ET-1, NOS2 and EGFR in different groups (×100). OD values of ( B ) ET-1, ( C ) NOS2 and ( D ) EGFR (Compared with normal group, ## p

    Techniques Used: Immunohistochemistry

    32) Product Images from "DACH1 suppresses breast cancer as a negative regulator of CD44"

    Article Title: DACH1 suppresses breast cancer as a negative regulator of CD44

    Journal: Scientific Reports

    doi: 10.1038/s41598-017-04709-2

    DACH1 reduced the expression of CD44, Myc, Sox2, Fibronectin, Vimentin, EGFR and Ki-67 in vivo . Both representative immunohistochemistry images and scoring results showed that overexpression of DACH1 ( a ) down-regulated the protein abundance of CD44 ( b ), Myc ( c ), Sox2 ( d ), Fibronectin ( e ), Vimentin ( f ), EGFR ( g ) and Ki-67 ( h ) in nude mice xenograft tumors.
    Figure Legend Snippet: DACH1 reduced the expression of CD44, Myc, Sox2, Fibronectin, Vimentin, EGFR and Ki-67 in vivo . Both representative immunohistochemistry images and scoring results showed that overexpression of DACH1 ( a ) down-regulated the protein abundance of CD44 ( b ), Myc ( c ), Sox2 ( d ), Fibronectin ( e ), Vimentin ( f ), EGFR ( g ) and Ki-67 ( h ) in nude mice xenograft tumors.

    Techniques Used: Expressing, In Vivo, Immunohistochemistry, Over Expression, Mouse Assay

    The expression of DACH1 and CD44 correlated with VIM , FN1 , YBX1 , FOXA1 , EGFR and MKI67 in breast cancer tissues. Correlation analysis of public dataset GSE 20685 showed that DACH1 was inversely correlated with cancer stem cell marker CD44 ( a ), mesenchymal markers FN1 ( b ) and VIM ( c ) as well as basal-like markers EGFR ( e ) and MKI67 ( f ), while positively associated with luminal marker FOXA1 ( d ). CD44 was parallel with FN1 ( g ) and VIM ( h ), YBX1 ( i ), EGFR ( k ) and MKI67 ( l ), while negatively associated with FOXA1 ( j ).
    Figure Legend Snippet: The expression of DACH1 and CD44 correlated with VIM , FN1 , YBX1 , FOXA1 , EGFR and MKI67 in breast cancer tissues. Correlation analysis of public dataset GSE 20685 showed that DACH1 was inversely correlated with cancer stem cell marker CD44 ( a ), mesenchymal markers FN1 ( b ) and VIM ( c ) as well as basal-like markers EGFR ( e ) and MKI67 ( f ), while positively associated with luminal marker FOXA1 ( d ). CD44 was parallel with FN1 ( g ) and VIM ( h ), YBX1 ( i ), EGFR ( k ) and MKI67 ( l ), while negatively associated with FOXA1 ( j ).

    Techniques Used: Expressing, Marker

    The association between the expression of DACH1 and CD44 with VIM , FN1 , YBX1 , FOXA1 , EGFR and MKI67 in breast cancer cell lines. Correlation analysis of data with a total of 51 breast cancer cell lines showed that DACH1 was inversely correlated with CD44 ( a ), FN1 ( b ), VIM ( c ), EGFR ( e ) and MKI67 ( f ), while positively associated with FOXA1 ( d ). In contrast, CD44 was parallel with FN1 ( g ), VIM ( h ), YBX1 ( i ), EGFR ( k ) and MKI67 ( l ), but negatively related to FOXA1 ( j ).
    Figure Legend Snippet: The association between the expression of DACH1 and CD44 with VIM , FN1 , YBX1 , FOXA1 , EGFR and MKI67 in breast cancer cell lines. Correlation analysis of data with a total of 51 breast cancer cell lines showed that DACH1 was inversely correlated with CD44 ( a ), FN1 ( b ), VIM ( c ), EGFR ( e ) and MKI67 ( f ), while positively associated with FOXA1 ( d ). In contrast, CD44 was parallel with FN1 ( g ), VIM ( h ), YBX1 ( i ), EGFR ( k ) and MKI67 ( l ), but negatively related to FOXA1 ( j ).

    Techniques Used: Expressing

    33) Product Images from "The interplay between AR, EGF receptor and MMP-9 signaling pathways in invasive prostate cancer"

    Article Title: The interplay between AR, EGF receptor and MMP-9 signaling pathways in invasive prostate cancer

    Journal: Molecular Medicine

    doi: 10.1186/s10020-018-0035-4

    The effect of overexpression of MMP9 on the expression of EGFR, AR, p-GSK-3β and p27 in VCaP cells. a Immunoblot analysis was performed to examine the expression of EGFR in VCaP cells that were transfected with pLX-control vector (PLX-Ctrl) or pLX-MMP9 vector (PLX-MMP9). b Expression of AR in VCaP cells that were transfected with pLX-control vector (PLX-Ctrl) or pLX-MMP9 vector (PLX-MMP9). c and d Expression of p-GSK-3β and p27 in VCaP cells that were transfected with pLX-control vector (PLX-Ctrl) or pLX-MMP9 vector (PLX-MMP9). Data presented is average of two independent experiments (±SD). p
    Figure Legend Snippet: The effect of overexpression of MMP9 on the expression of EGFR, AR, p-GSK-3β and p27 in VCaP cells. a Immunoblot analysis was performed to examine the expression of EGFR in VCaP cells that were transfected with pLX-control vector (PLX-Ctrl) or pLX-MMP9 vector (PLX-MMP9). b Expression of AR in VCaP cells that were transfected with pLX-control vector (PLX-Ctrl) or pLX-MMP9 vector (PLX-MMP9). c and d Expression of p-GSK-3β and p27 in VCaP cells that were transfected with pLX-control vector (PLX-Ctrl) or pLX-MMP9 vector (PLX-MMP9). Data presented is average of two independent experiments (±SD). p

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

    The effect of inhibition of the PI3K/AKT/AR axis on the expression of AR, MMP-9 and EGFR in PC-3 cells. a Immunoblot analysis on the expression of AR in PC-3 cells that were transfected with pCMV control or pCMV-AR vectors followed by treatment with PIP5K1α/AKT inhibitor ISA-2011B. b Immunoblot analysis on the expression of MMP-9 in PC-3 cells that were transfected with pCMV control vector followed by treatment with ISA-2011B. c Immunoblot analysis on the expression of MMP-9 in PC-3 cells that were transfected with pCMV-AR vector followed by treatment with ISA-2011B. d Immunoblot analysis on the expression of EGFR in PC-3 cells that were transfected with pCMV control or pCMV-AR vectors followed by treatment with ISA-2011B. Data presented is the average of at least two independent experiments (±SD). p
    Figure Legend Snippet: The effect of inhibition of the PI3K/AKT/AR axis on the expression of AR, MMP-9 and EGFR in PC-3 cells. a Immunoblot analysis on the expression of AR in PC-3 cells that were transfected with pCMV control or pCMV-AR vectors followed by treatment with PIP5K1α/AKT inhibitor ISA-2011B. b Immunoblot analysis on the expression of MMP-9 in PC-3 cells that were transfected with pCMV control vector followed by treatment with ISA-2011B. c Immunoblot analysis on the expression of MMP-9 in PC-3 cells that were transfected with pCMV-AR vector followed by treatment with ISA-2011B. d Immunoblot analysis on the expression of EGFR in PC-3 cells that were transfected with pCMV control or pCMV-AR vectors followed by treatment with ISA-2011B. Data presented is the average of at least two independent experiments (±SD). p

    Techniques Used: Inhibition, Expressing, Transfection, Plasmid Preparation

    Evaluation of the effect of overexpression of AR in the presence or absence of DHT treatment on MMP-9 and EGFR expression in PCa cells. a Immunoblot analysis was performed to examine the expression of MMP-9 in VCaP cells that were transfected with pCMV control or pCMV-AR vectors followed by treatment with DHT or vehicle control. b Immunoblot analysis on the expression of AR in PC-3 cells that were transfected with pCMV control or pCMV-AR vectors followed by treatment with DHT. c Immunoblot analysis on the expression of MMP-9 in PC-3 cells that were transfected with pCMV control or pCMV-AR vectors followed by treatment with DHT. d Immunoblot analysis on the expression of EGFR in PC-3 cells that were transfected with pCMV control or pCMV-AR vectors followed by treatment with DHT. Data presented is the average of at least two independent experiments (±SD). p
    Figure Legend Snippet: Evaluation of the effect of overexpression of AR in the presence or absence of DHT treatment on MMP-9 and EGFR expression in PCa cells. a Immunoblot analysis was performed to examine the expression of MMP-9 in VCaP cells that were transfected with pCMV control or pCMV-AR vectors followed by treatment with DHT or vehicle control. b Immunoblot analysis on the expression of AR in PC-3 cells that were transfected with pCMV control or pCMV-AR vectors followed by treatment with DHT. c Immunoblot analysis on the expression of MMP-9 in PC-3 cells that were transfected with pCMV control or pCMV-AR vectors followed by treatment with DHT. d Immunoblot analysis on the expression of EGFR in PC-3 cells that were transfected with pCMV control or pCMV-AR vectors followed by treatment with DHT. Data presented is the average of at least two independent experiments (±SD). p

    Techniques Used: Over Expression, Expressing, Transfection

    Evaluation the effect of overexpression of AR and DHT treatment on EGFR-related downstream effectors of AKT. a Immunoblot analysis was performed to examine the expression of p-GSK-3β in VCaP cells that were transfected with pCMV control or pCMV-AR vectors followed by treatment with DHT or vehicle control. b . Immunoblot analysis was performed to examine the expression of p27 in VCaP cells that were transfected with pCMV control or pCMV-AR vectors followed by treatment with DHT or vehicle control. Antibody against GAPDH was used as loading control. Data presented is average of three independent experiments (±SD). p
    Figure Legend Snippet: Evaluation the effect of overexpression of AR and DHT treatment on EGFR-related downstream effectors of AKT. a Immunoblot analysis was performed to examine the expression of p-GSK-3β in VCaP cells that were transfected with pCMV control or pCMV-AR vectors followed by treatment with DHT or vehicle control. b . Immunoblot analysis was performed to examine the expression of p27 in VCaP cells that were transfected with pCMV control or pCMV-AR vectors followed by treatment with DHT or vehicle control. Antibody against GAPDH was used as loading control. Data presented is average of three independent experiments (±SD). p

    Techniques Used: Over Expression, Expressing, Transfection

    34) Product Images from "Epidermal growth factor receptor promotes glioma progression by regulating xCT and GluN2B‐containing N‐methyl‐d‐aspartate–sensitive glutamate receptor signaling, et al. Epidermal growth factor receptor promotes glioma progression by regulating xCT and GluN2B‐containing N‐methyl‐d‐aspartate–sensitive glutamate receptor signaling"

    Article Title: Epidermal growth factor receptor promotes glioma progression by regulating xCT and GluN2B‐containing N‐methyl‐d‐aspartate–sensitive glutamate receptor signaling, et al. Epidermal growth factor receptor promotes glioma progression by regulating xCT and GluN2B‐containing N‐methyl‐d‐aspartate–sensitive glutamate receptor signaling

    Journal: Cancer Science

    doi: 10.1111/cas.13826

    Epidermal growth factor receptor ( EGFR ) interacts with and phosphorylates GluN2B in glioma cells. A, Immunofluorescence analysis of EGFR (red) and tyrosine‐phosphorylated GluN2B ( pG luN2B, green) in U87 MG ‐E cells that had been incubated in the presence of gefitinib (2 μmol/L) or DMSO vehicle for 2 hours. Nuclei were stained with Hoechst 33342 (blue). Scale bars, 20 μm. Arrowheads indicate colocalization of EGFR and phosphorylated GluN2B at the cell surface. B, U87 MG ‐E or T98G cells that had been incubated in the absence or presence of epidermal growth factor ( EGF) (20 ng/mL) or gefitinib (2 μmol/L) for 20 minutes were lysed and subjected to immunoprecipitation ( IP ) with antibodies to EGFR or with control IgG. The resulting precipitates, as well as the original cell lysates (Input), were subjected to immunoblot analysis with antibodies to phospho‐GluN2B (Y1474), to GluN2B and to EGFR . C, GST fusion protein containing the catalytic domain of EGFR was assayed for kinase activity in vitro with GST or GST fusion proteins containing WT or Y1474F mutant forms of the COOH ‐terminal region of GluN2B. The reaction mixtures were subjected to SDS ‐ PAGE followed by staining with Ponceau S or by immunoblot analysis with antibodies to phosphotyrosine
    Figure Legend Snippet: Epidermal growth factor receptor ( EGFR ) interacts with and phosphorylates GluN2B in glioma cells. A, Immunofluorescence analysis of EGFR (red) and tyrosine‐phosphorylated GluN2B ( pG luN2B, green) in U87 MG ‐E cells that had been incubated in the presence of gefitinib (2 μmol/L) or DMSO vehicle for 2 hours. Nuclei were stained with Hoechst 33342 (blue). Scale bars, 20 μm. Arrowheads indicate colocalization of EGFR and phosphorylated GluN2B at the cell surface. B, U87 MG ‐E or T98G cells that had been incubated in the absence or presence of epidermal growth factor ( EGF) (20 ng/mL) or gefitinib (2 μmol/L) for 20 minutes were lysed and subjected to immunoprecipitation ( IP ) with antibodies to EGFR or with control IgG. The resulting precipitates, as well as the original cell lysates (Input), were subjected to immunoblot analysis with antibodies to phospho‐GluN2B (Y1474), to GluN2B and to EGFR . C, GST fusion protein containing the catalytic domain of EGFR was assayed for kinase activity in vitro with GST or GST fusion proteins containing WT or Y1474F mutant forms of the COOH ‐terminal region of GluN2B. The reaction mixtures were subjected to SDS ‐ PAGE followed by staining with Ponceau S or by immunoblot analysis with antibodies to phosphotyrosine

    Techniques Used: Immunofluorescence, Incubation, Staining, Immunoprecipitation, Activity Assay, In Vitro, Mutagenesis, SDS Page

    35) Product Images from "Radiotherapy Upregulates Programmed Death Ligand-1 through the Pathways Downstream of Epidermal Growth Factor Receptor in Glioma"

    Article Title: Radiotherapy Upregulates Programmed Death Ligand-1 through the Pathways Downstream of Epidermal Growth Factor Receptor in Glioma

    Journal: EBioMedicine

    doi: 10.1016/j.ebiom.2018.01.027

    IHC staining for PD-L1 and EGFR in glioma specimens. Representative images of HE staining, positive PD-L1 and EGFR staining on slides from a selected tumor specimen, and representative HE staining, negative PD-L1 and EGFR staining on slides from another tumor specimen, are shown. Three consecutive slices from the same patient were stained, and one was chosen as representative image for shown.
    Figure Legend Snippet: IHC staining for PD-L1 and EGFR in glioma specimens. Representative images of HE staining, positive PD-L1 and EGFR staining on slides from a selected tumor specimen, and representative HE staining, negative PD-L1 and EGFR staining on slides from another tumor specimen, are shown. Three consecutive slices from the same patient were stained, and one was chosen as representative image for shown.

    Techniques Used: Immunohistochemistry, Staining

    EGFR inhibitor abrogates the up-regulation of PD-L1 at the mRNA level caused by RT in glioma cells. Cell lines (U87 and U251) were treated with vehicle control, RT (5 or 10 Gy), or RT combined with AG490 (10 μM) for 24, 48 or 72 h. PD-L1 expression at the mRNA level was determined by RT-PCR and expressed as fold change relative to the vehicle group. Each column is shown as the means of three separate experiments; bars, SD. ANOVA; *** P
    Figure Legend Snippet: EGFR inhibitor abrogates the up-regulation of PD-L1 at the mRNA level caused by RT in glioma cells. Cell lines (U87 and U251) were treated with vehicle control, RT (5 or 10 Gy), or RT combined with AG490 (10 μM) for 24, 48 or 72 h. PD-L1 expression at the mRNA level was determined by RT-PCR and expressed as fold change relative to the vehicle group. Each column is shown as the means of three separate experiments; bars, SD. ANOVA; *** P

    Techniques Used: Expressing, Reverse Transcription Polymerase Chain Reaction

    RT activates the EGFR signaling and up-regulates the PD-L1 expression in glioma cells. The EGFR signaling and PD-L1 protein level in U87 and U251 cell lines were detected at 24, 48 and 72 h after 5 or 10 Gy irradiation. Representative images and quantitative data are shown. Each column is shown as the means of three separate experiments; bars, SD. ANOVA; * P
    Figure Legend Snippet: RT activates the EGFR signaling and up-regulates the PD-L1 expression in glioma cells. The EGFR signaling and PD-L1 protein level in U87 and U251 cell lines were detected at 24, 48 and 72 h after 5 or 10 Gy irradiation. Representative images and quantitative data are shown. Each column is shown as the means of three separate experiments; bars, SD. ANOVA; * P

    Techniques Used: Expressing, Irradiation

    EGFR inhibitor abrogates the up-regulation of PD-L1 caused by RT in glioma cells. EGFR signaling and PD-L1 protein level in U87 and U251 cell lines were detected at 24, 48 and 72 h after 5 or 10 Gy irradiation. Before irradiation, U87 and U251 cells were incubated in the presence of 10 μM AG490 at 37 °C for 1 h. Each column is shown as the means of three separate experiments; bars, SD. ANOVA; * P
    Figure Legend Snippet: EGFR inhibitor abrogates the up-regulation of PD-L1 caused by RT in glioma cells. EGFR signaling and PD-L1 protein level in U87 and U251 cell lines were detected at 24, 48 and 72 h after 5 or 10 Gy irradiation. Before irradiation, U87 and U251 cells were incubated in the presence of 10 μM AG490 at 37 °C for 1 h. Each column is shown as the means of three separate experiments; bars, SD. ANOVA; * P

    Techniques Used: Irradiation, Incubation

    36) Product Images from "LncRNA SLCO4A1-AS1 predicts poor prognosis and promotes proliferation and metastasis via the EGFR/MAPK pathway in colorectal cancer"

    Article Title: LncRNA SLCO4A1-AS1 predicts poor prognosis and promotes proliferation and metastasis via the EGFR/MAPK pathway in colorectal cancer

    Journal: International Journal of Biological Sciences

    doi: 10.7150/ijbs.38041

    Proposed schematic model illustrating the role of SLCO4A1-AS1 in regulating CRC by EGFR/MAPK signaling pathway. SLCO4A1-AS1 influences EGFR/MAPK signaling pathway by promoting the expression of EGFR, KRAS, BRAF, MEK, ERK, MAP3K1 and its corresponding phosphorylated protein levels, which further affect the proliferation, migration and invasion of CRC cells.
    Figure Legend Snippet: Proposed schematic model illustrating the role of SLCO4A1-AS1 in regulating CRC by EGFR/MAPK signaling pathway. SLCO4A1-AS1 influences EGFR/MAPK signaling pathway by promoting the expression of EGFR, KRAS, BRAF, MEK, ERK, MAP3K1 and its corresponding phosphorylated protein levels, which further affect the proliferation, migration and invasion of CRC cells.

    Techniques Used: Expressing, Migration

    37) Product Images from "Interaction of glycosphingolipids GD3 and GD2 with growth factor receptors maintains breast cancer stem cell phenotype"

    Article Title: Interaction of glycosphingolipids GD3 and GD2 with growth factor receptors maintains breast cancer stem cell phenotype

    Journal: Oncotarget

    doi: 10.18632/oncotarget.17665

    Association of GD2 and GD3 with GFRs in breast CSCs Association between GD2/GD3 and GFRs was evaluated using Twist-induced EMT of HMLE-Twist-ER cells (breast CSCs). ( A ) Cell lysates of breast CSCs were immunoprecipitated (IP) with mouse anti-GD2 mAb, anti-GD3 mAb, or normal mouse IgG, followed by Western blotting (WB). WB was probed with anti-EGFR (left panel), anti-c-Met (middle panel), or anti-integrin β1 (right panel) mAb to detect components of Ab-absorbed complexes. Integrin β1/GD2 association was used as positive control. ( B ) Semi-confluent monolayers of breast CSCs were fixed and permeabilized. Cells were immune-stained with anti-GD3 (red) and anti-EGFR (green) (upper panels) or with anti-GD2 (red) and anti-c-Met (green) (lower panels), for immunofluorescence double labeling in combination with DAPI staining (blue) for cell nuclei. Scale bar = 10 μm. ( C ) GD3/EGFR and GD2/c-Met associations were evaluated by immunogold-TEM. Gangliosides (GD2, GD3) were probed with 12 nm colloidal gold particles; GFRs (EGFR, c-Met) were probed with 6 nm colloidal gold particles. Scale bar = 100 or 25 nm. Regions indicated by white boxes are shown at higher magnification in lower panels. Scale bar = 100 or 25 nm. ( D ) GD3/EGFR and GD2/c-Met associations in breast CSCs were investigated by in situ proximity ligation assay (PLA). Each PLA signal is visualized as a red fluorescent spot, and represents one detected association event. Cell nuclei were stained with DAPI (blue). Scale bar = 20 μm.
    Figure Legend Snippet: Association of GD2 and GD3 with GFRs in breast CSCs Association between GD2/GD3 and GFRs was evaluated using Twist-induced EMT of HMLE-Twist-ER cells (breast CSCs). ( A ) Cell lysates of breast CSCs were immunoprecipitated (IP) with mouse anti-GD2 mAb, anti-GD3 mAb, or normal mouse IgG, followed by Western blotting (WB). WB was probed with anti-EGFR (left panel), anti-c-Met (middle panel), or anti-integrin β1 (right panel) mAb to detect components of Ab-absorbed complexes. Integrin β1/GD2 association was used as positive control. ( B ) Semi-confluent monolayers of breast CSCs were fixed and permeabilized. Cells were immune-stained with anti-GD3 (red) and anti-EGFR (green) (upper panels) or with anti-GD2 (red) and anti-c-Met (green) (lower panels), for immunofluorescence double labeling in combination with DAPI staining (blue) for cell nuclei. Scale bar = 10 μm. ( C ) GD3/EGFR and GD2/c-Met associations were evaluated by immunogold-TEM. Gangliosides (GD2, GD3) were probed with 12 nm colloidal gold particles; GFRs (EGFR, c-Met) were probed with 6 nm colloidal gold particles. Scale bar = 100 or 25 nm. Regions indicated by white boxes are shown at higher magnification in lower panels. Scale bar = 100 or 25 nm. ( D ) GD3/EGFR and GD2/c-Met associations in breast CSCs were investigated by in situ proximity ligation assay (PLA). Each PLA signal is visualized as a red fluorescent spot, and represents one detected association event. Cell nuclei were stained with DAPI (blue). Scale bar = 20 μm.

    Techniques Used: Immunoprecipitation, Western Blot, Positive Control, Staining, Immunofluorescence, Labeling, Transmission Electron Microscopy, In Situ, Proximity Ligation Assay

    38) Product Images from "IRF6 is a mediator of Notch pro-differentiation and tumour suppressive function in keratinocytes"

    Article Title: IRF6 is a mediator of Notch pro-differentiation and tumour suppressive function in keratinocytes

    Journal: The EMBO Journal

    doi: 10.1038/emboj.2011.325

    Opposite pattern of Notch1 and IRF6 versus EGFR and IRF7 expression in human SCCs. ( A ) RNA samples from a panel of freshly excised cutaneous SCCs and normal epidermis were analysed by real-time RT–PCR for the indicated genes. Values are expressed
    Figure Legend Snippet: Opposite pattern of Notch1 and IRF6 versus EGFR and IRF7 expression in human SCCs. ( A ) RNA samples from a panel of freshly excised cutaneous SCCs and normal epidermis were analysed by real-time RT–PCR for the indicated genes. Values are expressed

    Techniques Used: Expressing, Quantitative RT-PCR

    39) Product Images from "Capsaicin and sorafenib combination treatment exerts synergistic anti-hepatocellular carcinoma activity by suppressing EGFR and PI3K/Akt/mTOR signaling"

    Article Title: Capsaicin and sorafenib combination treatment exerts synergistic anti-hepatocellular carcinoma activity by suppressing EGFR and PI3K/Akt/mTOR signaling

    Journal: Oncology Reports

    doi: 10.3892/or.2018.6754

    Capsaicin and sorafenib inhibit HCC cancer cell growth, invasion and metastasis synergistically in vivo . (A) 1×10 7 LM3 cells were inoculated into BALB/c-nude mice. The mice were randomized into 4 groups (n=6) and treated with PBS (with DMSO), capsaicin (Cap), sorafenib (Sora) and their combination daily for 28 days. Tumor volumes and body weight were measured every 2 days. (B) The tumors were excised after the last treatment. EGFR, epidermal growth factor receptor; PBS, phosphate-buffered saline; DMSO, dimethyl sulfoxide. Capsaicin and sorafenib inhibit HCC cancer cell growth, invasion and metastasis synergistically in vivo . (C) The apoptosis, autophagy, invasion, metastasis and phosphorylation of Ki67, Bax, P62, matrix metalloproteinase (MMP)2, MMP9, P-Akt and P-EGFR in xenograft tumor tissues were detected by immunohistochemistry (magnification, ×400). Con, control; Cap, capsaicin; Sora, sorafenib. Capsaicin and sorafenib inhibit HCC cancer cell growth, invasion and metastasis synergistically in vivo . (D) The liver and kidney biochemical functions were evaluated. The AST, ALT, BUN, CR and BUN/CR levels of were detected in mouse blood by ELISA. (E) The liver and kidneys from the control and the three treatment groups were stained with hematoxylin and eosin to evaluate the toxicity after treatment. The histological structures of the liver and kidney were observed and compared microscopically (magnification, ×200). AST, aspartate aminotransferase; ALT, alanine aminotransferase; BUN, blood urea nitrogen; CR, creatinine; Con, control; Cap, capsaicin; Sora, sorafenib.
    Figure Legend Snippet: Capsaicin and sorafenib inhibit HCC cancer cell growth, invasion and metastasis synergistically in vivo . (A) 1×10 7 LM3 cells were inoculated into BALB/c-nude mice. The mice were randomized into 4 groups (n=6) and treated with PBS (with DMSO), capsaicin (Cap), sorafenib (Sora) and their combination daily for 28 days. Tumor volumes and body weight were measured every 2 days. (B) The tumors were excised after the last treatment. EGFR, epidermal growth factor receptor; PBS, phosphate-buffered saline; DMSO, dimethyl sulfoxide. Capsaicin and sorafenib inhibit HCC cancer cell growth, invasion and metastasis synergistically in vivo . (C) The apoptosis, autophagy, invasion, metastasis and phosphorylation of Ki67, Bax, P62, matrix metalloproteinase (MMP)2, MMP9, P-Akt and P-EGFR in xenograft tumor tissues were detected by immunohistochemistry (magnification, ×400). Con, control; Cap, capsaicin; Sora, sorafenib. Capsaicin and sorafenib inhibit HCC cancer cell growth, invasion and metastasis synergistically in vivo . (D) The liver and kidney biochemical functions were evaluated. The AST, ALT, BUN, CR and BUN/CR levels of were detected in mouse blood by ELISA. (E) The liver and kidneys from the control and the three treatment groups were stained with hematoxylin and eosin to evaluate the toxicity after treatment. The histological structures of the liver and kidney were observed and compared microscopically (magnification, ×200). AST, aspartate aminotransferase; ALT, alanine aminotransferase; BUN, blood urea nitrogen; CR, creatinine; Con, control; Cap, capsaicin; Sora, sorafenib.

    Techniques Used: In Vivo, Mouse Assay, Immunohistochemistry, AST Assay, Enzyme-linked Immunosorbent Assay, Staining

    Capsaicin and sorafenib reduce the expression of EGFR, PI3K/Akt/mTOR pathway components. Capsaicin and sorafenib downregulate EGFR, PI3K/Akt/mTOR signaling and the client protein P-p70s6k in LM3 cells. The combination of capsaicin and sorafenib exerted a synergistic effect on this pathway. Cells were serum-starved for 24 h and then treated with capsaicin, sorafenib and their combination for 12 h. Protein lysates were analyzed by western blotting with the indicated antibodies. GAPDH served as a loading control. Values represented the mean ± standard error of the mean from three independent experiments. #, Capsaicin vs. combination; +, sorafenib vs combination. ++ and ###/+++, P
    Figure Legend Snippet: Capsaicin and sorafenib reduce the expression of EGFR, PI3K/Akt/mTOR pathway components. Capsaicin and sorafenib downregulate EGFR, PI3K/Akt/mTOR signaling and the client protein P-p70s6k in LM3 cells. The combination of capsaicin and sorafenib exerted a synergistic effect on this pathway. Cells were serum-starved for 24 h and then treated with capsaicin, sorafenib and their combination for 12 h. Protein lysates were analyzed by western blotting with the indicated antibodies. GAPDH served as a loading control. Values represented the mean ± standard error of the mean from three independent experiments. #, Capsaicin vs. combination; +, sorafenib vs combination. ++ and ###/+++, P

    Techniques Used: Expressing, Western Blot

    40) Product Images from "Regulatory mechanisms of betacellulin in CXCL8 production from lung cancer cells"

    Article Title: Regulatory mechanisms of betacellulin in CXCL8 production from lung cancer cells

    Journal: Journal of Translational Medicine

    doi: 10.1186/1479-5876-12-70

    Expression of ErbB family receptors in A549 human lung tumor cell line. A549 cell line is positive for EGFR (red labeling) (A) , and ErbB2 (green) (B) and negative for ErbB3 (C) and ErbB4 (D) (FITC staining, green), respectively, in immunofluorescence analysis. DAPI staining (blue) indicates the localization of nuclei. One representative image from three independent experiments is presented.
    Figure Legend Snippet: Expression of ErbB family receptors in A549 human lung tumor cell line. A549 cell line is positive for EGFR (red labeling) (A) , and ErbB2 (green) (B) and negative for ErbB3 (C) and ErbB4 (D) (FITC staining, green), respectively, in immunofluorescence analysis. DAPI staining (blue) indicates the localization of nuclei. One representative image from three independent experiments is presented.

    Techniques Used: Expressing, Labeling, Staining, Immunofluorescence

    Related Articles

    Staining:

    Article Title: cIAP1 regulates the EGFR/Snai2 axis in triple-negative breast cancer cells
    Article Snippet: .. For PLA assay, cover slips were stained with anti EGFR (mouse, dilution 1:200, abcam) and/or cIAP1 (rabbit, dilution 1:2000, abcam). .. After the overnight incubation with primary antibodies, cells were stained with the PLA probes diluted 1:5 in antibody diluent (Sigma-Aldrich) in a humidified chamber for 1 h at 37 °C.

    Incubation:

    Article Title: cIAP1 regulates the EGFR/Snai2 axis in triple-negative breast cancer cells
    Article Snippet: .. Cover slips were then incubated overnight at 4 °C in a humidified chamber with antibodies directed to EGFR (mouse, dilution 1:200, abcam). .. Fluorescence images were acquired using a fluorescence microscopy and digital image acquisition on a Nikon Eclipse E1000 equipped with a DSU3 CCD camera.

    Proximity Ligation Assay:

    Article Title: cIAP1 regulates the EGFR/Snai2 axis in triple-negative breast cancer cells
    Article Snippet: .. For PLA assay, cover slips were stained with anti EGFR (mouse, dilution 1:200, abcam) and/or cIAP1 (rabbit, dilution 1:2000, abcam). .. After the overnight incubation with primary antibodies, cells were stained with the PLA probes diluted 1:5 in antibody diluent (Sigma-Aldrich) in a humidified chamber for 1 h at 37 °C.

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  • 94
    Abcam rabbit anti egfr
    Effects of the upregulation or downregulation of <t>RBM10</t> expression on <t>EGFR</t> expression. (A) Effects of RBM10 expression on EGFR expression in A549 cells examined by immunofluorescence assay; magnification, ×400. (B) Effects of RBM10 expression on EGFR expression in H1299 cells examined by immunofluorescence assay; magnification, ×400. (C) Effects of RBM10 expression on EGFR expression in A549 cells examined by western blot analysis and quantitative analysis of the western blots. (D) Effect of RBM10 expression on EGFR expression in H1299 cells examined by western blot analysis and quantitative analysis of the western blots. # P
    Rabbit Anti Egfr, supplied by Abcam, used in various techniques. Bioz Stars score: 94/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    egfr  (Abcam)
    93
    Abcam egfr
    Silencing of <t>cIAP1</t> reduces <t>EGFR</t> levels. a BT549 and b MCF10A cells were transfected with control or cIAP1-specific siRNAs before being serum-starved overnight, stimulated with 20 ng/ml EGF and analyzed by western blot to detect the indicate proteins. c BT549 cells were transfected with control or cIAP1-specific siRNAs and, after 48 h, cells were serum-starved for 24 h and then stimulated 30 min with 20 ng/ml EGF. Fixed cells were incubated with anti-EGFR antibody and nuclei stained with DAPI. d LRIG1 expression levels were evaluated by real-time PCR in BT549 cells serum-starved and stimulated with 20 ng/ml EGF after transfection with control or cIAP1-directed siRNAs. Unstimulated siCtr vs. sicIAP1 * P = 0.0175, EGF 3 h siCtr vs. sicIAP1 * P = 0.0341; n = 3; two-tailed paired t- test. e Levels of Snai2 and LRIG1 in BT549 cells silenced for cIAP1 and stimulated or not with 20 ng/ml EGF. f EGFR levels in BT549 cells silenced for cIAP1 and LRIG1, stimulated or not with 20 ng/ml EGF. g LRIG1 expression levels were evaluated by real-time PCR in MCF10A cells serum-starved and stimulated with 20 ng/ml EGF after transfection with control or cIAP1-directed siRNAs. ns not significant; n = 4; two-tailed paired t- test
    Egfr, supplied by Abcam, used in various techniques. Bioz Stars score: 93/100, based on 89 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    88
    Abcam egfr p egfr
    Down-regulation and mutation of the PTEN gene, as well as up-regulated phosphorylation of AKT and IGF-1R proteins, in trastuzumab-resistant NCI-N87/TR cells. a . Western blotting showed the expression of Her-2, PTEN, <t>EGFR,</t> P-EGFR, IGF-1R, P-IGF-1R, AKT, P-AKT, survivin, CDK2, and p27kipl proteins in NCI-N87 cells and NCI-N87/TR cells. Actin or tubulin expression indicated equal loading. All the gels run under the same experimental conditions and the experiments were repeated 3 times. The representative images were cropped and shown. Data were presented as mean ± SD and analyzed using Student’s t-test. b . Expression of the Her-2 and PTEN genes in NCI-N87 cells and NCI-N87/TR cells shown by real-time PCR. The experiments were repeated 3 times. Data were presented as mean ± SD and analyzed using Student’s t-test. c . Results of PTEN exon 5 sequencing. Mutations at 1309 bp and 1392 bp were detected in NCI-N87/TR cells (A→G and G→A), with the former inducing (H→R; H: His, R: Arg) and the latter inducing (A→T; A: Ala, T: Thr). d . Results of PTEN exon7 sequencing. A mutation at 1732 bp was found in NCI-N87 cells (G→A), inducing (R→Q; R: Arg, Q: Gln), while a mutation at 1763 bp (T→A) was silent. e . Results of PTEN exon 8 sequencing. Mutations at 1955 bp and 1968 bp were found in NCI-N87/TR cells (T→C and A→G), with the former being silent and the latter inducing (K→E; K: Lys, E: Glu).
    Egfr P Egfr, supplied by Abcam, used in various techniques. Bioz Stars score: 88/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    Abcam rabbit monoclonal anti egfr
    Secretion of the immunodominant antigen is not required for immunodominance. (A) Schematic representation of the <t>B8-mCherry</t> fusion proteins; the location of the signal peptide, GGSGGS linker, TMD, and mCherry are depicted. (B) B8TMmC is not secreted. HeLa cells were infected at an MOI of 5 with B8TMmC or B8mC. Cells and supernatant were harvested at 4 hpi for subcellular fractionation and mCherry and <t>EGFR</t> expression was determined by western blot; equal loading and transfer of samples was confirmed with ponceau S red (P-Red) staining. CE = cytoplasmic extract; ME = membrane extract; SN = supernatant. Data are representative of two independent experiments. (C, D) Comparable CTL priming by B8TMmC and B8mC. CD8 + T cell responses in the spleen of B6 (n = 5) i.n. infected with 5 x 10 3 pfu (C) and 1.5 x 10 4 pfu (D) B8TMmC or B8mC were determined by ex vivo restimulation with CPXV peptides and ICS at 8 dpi. Data are representative of two independent experiments. (E) Cell-associated antigen is cross-presented more efficiently than soluble antigen. B8-specific CD8 + T cell responses in the spleen of B6 (n = 5) i.n. infected with 1.5 x 10 5 pfu B8TMmC or B8mC were determined by tetramer staining at 8, 9, and 10 dpi. Data are representative of two independent experiments. (F) CD8 + T cell responses require BATF3 + DCs. B6 and Batf3 -/- mice (n = 7–10) were i.n. infected with 5 x 10 3 pfu B8TMmC or B8mC and the B8-specific CD8 + T cell responses in the spleen were determined at 6 dpi. n = 3 mock-infected mice. Data are the combined results of three independent experiments.
    Rabbit Monoclonal Anti Egfr, supplied by Abcam, used in various techniques. Bioz Stars score: 91/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Effects of the upregulation or downregulation of RBM10 expression on EGFR expression. (A) Effects of RBM10 expression on EGFR expression in A549 cells examined by immunofluorescence assay; magnification, ×400. (B) Effects of RBM10 expression on EGFR expression in H1299 cells examined by immunofluorescence assay; magnification, ×400. (C) Effects of RBM10 expression on EGFR expression in A549 cells examined by western blot analysis and quantitative analysis of the western blots. (D) Effect of RBM10 expression on EGFR expression in H1299 cells examined by western blot analysis and quantitative analysis of the western blots. # P

    Journal: International Journal of Oncology

    Article Title: Functional role of RBM10 in lung adenocarcinoma proliferation

    doi: 10.3892/ijo.2018.4643

    Figure Lengend Snippet: Effects of the upregulation or downregulation of RBM10 expression on EGFR expression. (A) Effects of RBM10 expression on EGFR expression in A549 cells examined by immunofluorescence assay; magnification, ×400. (B) Effects of RBM10 expression on EGFR expression in H1299 cells examined by immunofluorescence assay; magnification, ×400. (C) Effects of RBM10 expression on EGFR expression in A549 cells examined by western blot analysis and quantitative analysis of the western blots. (D) Effect of RBM10 expression on EGFR expression in H1299 cells examined by western blot analysis and quantitative analysis of the western blots. # P

    Article Snippet: The antibodies used were as follows: Rabbit anti-RBM10 (ab72423, 1:2,000 dilution), rabbit anti-p53 (ab131442, 1:500 dilution), rabbit anti-EGFR (ab131498, 1:500 dilution), rabbit anti-Bax (ab182733, 1:1,000 dilution), rabbit anti-Bcl-2 (ab32124, 1:1,000 dilution), rabbit anti-caspase-8 (ab25901, 1:1,000 dilution) and rabbit anti-GAPDH (ab37168, 1:1,000 dilution) antibodies were obtained from Abcam (Cambridge, MA, USA).

    Techniques: Expressing, Immunofluorescence, Western Blot

    Silencing of cIAP1 reduces EGFR levels. a BT549 and b MCF10A cells were transfected with control or cIAP1-specific siRNAs before being serum-starved overnight, stimulated with 20 ng/ml EGF and analyzed by western blot to detect the indicate proteins. c BT549 cells were transfected with control or cIAP1-specific siRNAs and, after 48 h, cells were serum-starved for 24 h and then stimulated 30 min with 20 ng/ml EGF. Fixed cells were incubated with anti-EGFR antibody and nuclei stained with DAPI. d LRIG1 expression levels were evaluated by real-time PCR in BT549 cells serum-starved and stimulated with 20 ng/ml EGF after transfection with control or cIAP1-directed siRNAs. Unstimulated siCtr vs. sicIAP1 * P = 0.0175, EGF 3 h siCtr vs. sicIAP1 * P = 0.0341; n = 3; two-tailed paired t- test. e Levels of Snai2 and LRIG1 in BT549 cells silenced for cIAP1 and stimulated or not with 20 ng/ml EGF. f EGFR levels in BT549 cells silenced for cIAP1 and LRIG1, stimulated or not with 20 ng/ml EGF. g LRIG1 expression levels were evaluated by real-time PCR in MCF10A cells serum-starved and stimulated with 20 ng/ml EGF after transfection with control or cIAP1-directed siRNAs. ns not significant; n = 4; two-tailed paired t- test

    Journal: Cell Death and Differentiation

    Article Title: cIAP1 regulates the EGFR/Snai2 axis in triple-negative breast cancer cells

    doi: 10.1038/s41418-018-0100-0

    Figure Lengend Snippet: Silencing of cIAP1 reduces EGFR levels. a BT549 and b MCF10A cells were transfected with control or cIAP1-specific siRNAs before being serum-starved overnight, stimulated with 20 ng/ml EGF and analyzed by western blot to detect the indicate proteins. c BT549 cells were transfected with control or cIAP1-specific siRNAs and, after 48 h, cells were serum-starved for 24 h and then stimulated 30 min with 20 ng/ml EGF. Fixed cells were incubated with anti-EGFR antibody and nuclei stained with DAPI. d LRIG1 expression levels were evaluated by real-time PCR in BT549 cells serum-starved and stimulated with 20 ng/ml EGF after transfection with control or cIAP1-directed siRNAs. Unstimulated siCtr vs. sicIAP1 * P = 0.0175, EGF 3 h siCtr vs. sicIAP1 * P = 0.0341; n = 3; two-tailed paired t- test. e Levels of Snai2 and LRIG1 in BT549 cells silenced for cIAP1 and stimulated or not with 20 ng/ml EGF. f EGFR levels in BT549 cells silenced for cIAP1 and LRIG1, stimulated or not with 20 ng/ml EGF. g LRIG1 expression levels were evaluated by real-time PCR in MCF10A cells serum-starved and stimulated with 20 ng/ml EGF after transfection with control or cIAP1-directed siRNAs. ns not significant; n = 4; two-tailed paired t- test

    Article Snippet: For PLA assay, cover slips were stained with anti EGFR (mouse, dilution 1:200, abcam) and/or cIAP1 (rabbit, dilution 1:2000, abcam).

    Techniques: Transfection, Western Blot, Incubation, Staining, Expressing, Real-time Polymerase Chain Reaction, Two Tailed Test

    SM83 treatment results in anti-tumor and anti-metastasis effect. a for primary tumor volumes), formalin-fixed and paraffin-embedded, and stained with an anti-human vimentin antibody to detect spontaneous metastasis (bottom panel). b Number (untreated n = 7, SM83-treated mice n ; * P = 0.0238. Unpaired two-tailed t- test) and c size (35 metastases/group; * P = 0.0107. Unpaired two-tailed t- test) of spontaneous MDA-MB231 lung metastases were evaluated. d Western blot showing the levels of cIAP1, EGFR, ERK1/2, pERK1/2, and Snai2 in primary tumors at the end of the experiment described in Fig. 8a

    Journal: Cell Death and Differentiation

    Article Title: cIAP1 regulates the EGFR/Snai2 axis in triple-negative breast cancer cells

    doi: 10.1038/s41418-018-0100-0

    Figure Lengend Snippet: SM83 treatment results in anti-tumor and anti-metastasis effect. a for primary tumor volumes), formalin-fixed and paraffin-embedded, and stained with an anti-human vimentin antibody to detect spontaneous metastasis (bottom panel). b Number (untreated n = 7, SM83-treated mice n ; * P = 0.0238. Unpaired two-tailed t- test) and c size (35 metastases/group; * P = 0.0107. Unpaired two-tailed t- test) of spontaneous MDA-MB231 lung metastases were evaluated. d Western blot showing the levels of cIAP1, EGFR, ERK1/2, pERK1/2, and Snai2 in primary tumors at the end of the experiment described in Fig. 8a

    Article Snippet: For PLA assay, cover slips were stained with anti EGFR (mouse, dilution 1:200, abcam) and/or cIAP1 (rabbit, dilution 1:2000, abcam).

    Techniques: Staining, Mouse Assay, Two Tailed Test, Multiple Displacement Amplification, Western Blot

    cIAP1 reduces EGFR stability, but promotes its gene transcription. a EGFR and cIAP1 interaction was tested in BT549 cells using PLA assay. b BT549 cells stably expressing Myc/Flag-tagged EGFR were serum-starved and stimulated with 20 ng/ml EGF for the indicated times. Cells were lysed and EGFR was immunoprecipitated with anti-Flag antibody. Western blot was performed to evaluate the interaction of ectopic EGFR with cIAP1 and c-Cbl. c BT549 and MCF10A cells stably expressing Myc/Flag-tagged EGFR were serum-starved, pre-treated with 100 (BT549) or 50 (MCF10A) µg/ml cycloheximide for 30 min and stimulated with 20 ng/ml EGF for the indicated times. Levels of ectopic EGFR were detected with anti-Myc antibody. d Ectopic EGFR was immunoprecipitated as described in Fig. 6b from BT549 cells transfected with control and cIAP1-specific siRNAs. Western blot shows total levels of c-Cbl and the amount of c-Cbl interacting with EGFR. e BT549 and f MCF10A cell stably expressing ectopic Myc/Flag-tagged EGFR were further transduced with lentiviral particles to overexpress c-Cbl or GFP as a control. Cells were silenced for cIAP1, serum-starved, and stimulated with 20 ng/ml EGF as described above, and analyzed by western blot to evaluate the levels of ectopic EGFR by using a Myc-tagged-specific antibody. g BT549 (left panel) and MCF10A (right panel) were silenced for cIAP1 and analyzed by real-time PCR to quantify the levels of EGFR expression fold relative to GAPDH. BT549: unstimulated siCtr vs. sicIAP1 * P = 0.0134, EGF 3 h siCtr vs. sicIAP1 * P = 0.0270; n = 3. MCF10A: unstimulated siCtr vs. sicIAP1 *** P = 0.0004, EGF 3 h siCtr vs. sicIAP1 ** P = 0.0183; n = 4; two-tailed paired t- test

    Journal: Cell Death and Differentiation

    Article Title: cIAP1 regulates the EGFR/Snai2 axis in triple-negative breast cancer cells

    doi: 10.1038/s41418-018-0100-0

    Figure Lengend Snippet: cIAP1 reduces EGFR stability, but promotes its gene transcription. a EGFR and cIAP1 interaction was tested in BT549 cells using PLA assay. b BT549 cells stably expressing Myc/Flag-tagged EGFR were serum-starved and stimulated with 20 ng/ml EGF for the indicated times. Cells were lysed and EGFR was immunoprecipitated with anti-Flag antibody. Western blot was performed to evaluate the interaction of ectopic EGFR with cIAP1 and c-Cbl. c BT549 and MCF10A cells stably expressing Myc/Flag-tagged EGFR were serum-starved, pre-treated with 100 (BT549) or 50 (MCF10A) µg/ml cycloheximide for 30 min and stimulated with 20 ng/ml EGF for the indicated times. Levels of ectopic EGFR were detected with anti-Myc antibody. d Ectopic EGFR was immunoprecipitated as described in Fig. 6b from BT549 cells transfected with control and cIAP1-specific siRNAs. Western blot shows total levels of c-Cbl and the amount of c-Cbl interacting with EGFR. e BT549 and f MCF10A cell stably expressing ectopic Myc/Flag-tagged EGFR were further transduced with lentiviral particles to overexpress c-Cbl or GFP as a control. Cells were silenced for cIAP1, serum-starved, and stimulated with 20 ng/ml EGF as described above, and analyzed by western blot to evaluate the levels of ectopic EGFR by using a Myc-tagged-specific antibody. g BT549 (left panel) and MCF10A (right panel) were silenced for cIAP1 and analyzed by real-time PCR to quantify the levels of EGFR expression fold relative to GAPDH. BT549: unstimulated siCtr vs. sicIAP1 * P = 0.0134, EGF 3 h siCtr vs. sicIAP1 * P = 0.0270; n = 3. MCF10A: unstimulated siCtr vs. sicIAP1 *** P = 0.0004, EGF 3 h siCtr vs. sicIAP1 ** P = 0.0183; n = 4; two-tailed paired t- test

    Article Snippet: For PLA assay, cover slips were stained with anti EGFR (mouse, dilution 1:200, abcam) and/or cIAP1 (rabbit, dilution 1:2000, abcam).

    Techniques: Proximity Ligation Assay, Stable Transfection, Expressing, Immunoprecipitation, Western Blot, Transfection, Transduction, Real-time Polymerase Chain Reaction, Two Tailed Test

    Depletion of cIAP1 hinders EGFR-dependent expression of Snai2. a MDA-MB231 and b BT549 cells were transfected with control or cIAP1-specific siRNAs and, after 48 h, serum-starved overnight. Then, cells were stimulated for the indicated time points with 50 ng/ml and 20 ng/ml EGF, respectively. Levels of Snai2 and activated ERK1/2 were detected, together with cIAP1, to check the transfection efficiency. c BT549 and d MCF10A—wild-type or bearing mutated EGFR—cells were transfected as in Fig. 4a and stimulated with the indicated EGFR ligands (20 ng/ml) to evaluate the expression of Snai2 by western blot. e BT549 and f MCF10A cells were transfected and serum-starved as described before, stimulated 3 h with 20 ng/ml EGF and lysed to extract RNA. Real-time PCR was performed to evaluate Snai2 fold expression relative to GAPDH. BT549: * P = 0.0151, ** P = 0.0036; n = 3; MCF10A: unstimulated siCtr vs. sicIAP1 * P = 0.0290, EGF 3 h siCtr vs. sicIAP1 * P = 0.0330; n = 5; two-tailed paired t- test

    Journal: Cell Death and Differentiation

    Article Title: cIAP1 regulates the EGFR/Snai2 axis in triple-negative breast cancer cells

    doi: 10.1038/s41418-018-0100-0

    Figure Lengend Snippet: Depletion of cIAP1 hinders EGFR-dependent expression of Snai2. a MDA-MB231 and b BT549 cells were transfected with control or cIAP1-specific siRNAs and, after 48 h, serum-starved overnight. Then, cells were stimulated for the indicated time points with 50 ng/ml and 20 ng/ml EGF, respectively. Levels of Snai2 and activated ERK1/2 were detected, together with cIAP1, to check the transfection efficiency. c BT549 and d MCF10A—wild-type or bearing mutated EGFR—cells were transfected as in Fig. 4a and stimulated with the indicated EGFR ligands (20 ng/ml) to evaluate the expression of Snai2 by western blot. e BT549 and f MCF10A cells were transfected and serum-starved as described before, stimulated 3 h with 20 ng/ml EGF and lysed to extract RNA. Real-time PCR was performed to evaluate Snai2 fold expression relative to GAPDH. BT549: * P = 0.0151, ** P = 0.0036; n = 3; MCF10A: unstimulated siCtr vs. sicIAP1 * P = 0.0290, EGF 3 h siCtr vs. sicIAP1 * P = 0.0330; n = 5; two-tailed paired t- test

    Article Snippet: For PLA assay, cover slips were stained with anti EGFR (mouse, dilution 1:200, abcam) and/or cIAP1 (rabbit, dilution 1:2000, abcam).

    Techniques: Expressing, Multiple Displacement Amplification, Transfection, Western Blot, Real-time Polymerase Chain Reaction, Two Tailed Test

    EGFR promotes Snai2 expression in a MAPK-dependent manner. a MDA-MB231, BT549, and MDA-MB157 cells were harvested after treatment for 2 h with 10 μM inhibitor of PI3K (LY294002), AKT (Triciribine), MEK (UO126), and p38 (SB203580). Western blot was performed to analyze the levels of Snai2. b MDA-MB231 cells were transfected with siRNAs specific for cIAP1, ERK1, and ERK2 to detect the levels of Snai2 72 h after transfection. cIAP1 and ERK1/2 are shown to control the silencing efficiency. c ]. d For each cancer type available in the TCGA study, Spearman’s correlation between EGFR and SNAI2 was calculated using RNA-Seq data (expressed as log2 counts per million mapped reads). Only primary tumors were considered in the analysis. Red arrow indicates the correlation bar in breast cancers. e BT549 cells were serum-starved overnight and then stimulated with 20 ng/ml EGF and TGFα in time-course experiments. Snai2 levels are shown together with total and activated levels of EGFR. MDA-MB231 ( f ) and BT549 ( g ) cells were serum-starved overnight, pre-treated or not with 100 μg/ml cetuximab for 1 h and then stimulated with 20 ng/ml EGF for the indicated time points. Western blot was performed to detect Snai2 levels, total ERK1/2 and EGFR, and their activated levels. Values show the fold levels of Snai2 relative to untreated cells. h Human mammary epithelial cell lines, parental and bearing mutated EGFR, were serum-starved and stimulated with 20 ng/ml EGF for the indicated times to evaluate Snai2 levels

    Journal: Cell Death and Differentiation

    Article Title: cIAP1 regulates the EGFR/Snai2 axis in triple-negative breast cancer cells

    doi: 10.1038/s41418-018-0100-0

    Figure Lengend Snippet: EGFR promotes Snai2 expression in a MAPK-dependent manner. a MDA-MB231, BT549, and MDA-MB157 cells were harvested after treatment for 2 h with 10 μM inhibitor of PI3K (LY294002), AKT (Triciribine), MEK (UO126), and p38 (SB203580). Western blot was performed to analyze the levels of Snai2. b MDA-MB231 cells were transfected with siRNAs specific for cIAP1, ERK1, and ERK2 to detect the levels of Snai2 72 h after transfection. cIAP1 and ERK1/2 are shown to control the silencing efficiency. c ]. d For each cancer type available in the TCGA study, Spearman’s correlation between EGFR and SNAI2 was calculated using RNA-Seq data (expressed as log2 counts per million mapped reads). Only primary tumors were considered in the analysis. Red arrow indicates the correlation bar in breast cancers. e BT549 cells were serum-starved overnight and then stimulated with 20 ng/ml EGF and TGFα in time-course experiments. Snai2 levels are shown together with total and activated levels of EGFR. MDA-MB231 ( f ) and BT549 ( g ) cells were serum-starved overnight, pre-treated or not with 100 μg/ml cetuximab for 1 h and then stimulated with 20 ng/ml EGF for the indicated time points. Western blot was performed to detect Snai2 levels, total ERK1/2 and EGFR, and their activated levels. Values show the fold levels of Snai2 relative to untreated cells. h Human mammary epithelial cell lines, parental and bearing mutated EGFR, were serum-starved and stimulated with 20 ng/ml EGF for the indicated times to evaluate Snai2 levels

    Article Snippet: For PLA assay, cover slips were stained with anti EGFR (mouse, dilution 1:200, abcam) and/or cIAP1 (rabbit, dilution 1:2000, abcam).

    Techniques: Expressing, Multiple Displacement Amplification, Western Blot, Transfection, RNA Sequencing Assay

    Down-regulation and mutation of the PTEN gene, as well as up-regulated phosphorylation of AKT and IGF-1R proteins, in trastuzumab-resistant NCI-N87/TR cells. a . Western blotting showed the expression of Her-2, PTEN, EGFR, P-EGFR, IGF-1R, P-IGF-1R, AKT, P-AKT, survivin, CDK2, and p27kipl proteins in NCI-N87 cells and NCI-N87/TR cells. Actin or tubulin expression indicated equal loading. All the gels run under the same experimental conditions and the experiments were repeated 3 times. The representative images were cropped and shown. Data were presented as mean ± SD and analyzed using Student’s t-test. b . Expression of the Her-2 and PTEN genes in NCI-N87 cells and NCI-N87/TR cells shown by real-time PCR. The experiments were repeated 3 times. Data were presented as mean ± SD and analyzed using Student’s t-test. c . Results of PTEN exon 5 sequencing. Mutations at 1309 bp and 1392 bp were detected in NCI-N87/TR cells (A→G and G→A), with the former inducing (H→R; H: His, R: Arg) and the latter inducing (A→T; A: Ala, T: Thr). d . Results of PTEN exon7 sequencing. A mutation at 1732 bp was found in NCI-N87 cells (G→A), inducing (R→Q; R: Arg, Q: Gln), while a mutation at 1763 bp (T→A) was silent. e . Results of PTEN exon 8 sequencing. Mutations at 1955 bp and 1968 bp were found in NCI-N87/TR cells (T→C and A→G), with the former being silent and the latter inducing (K→E; K: Lys, E: Glu).

    Journal: Scientific Reports

    Article Title: Development of trastuzumab-resistant human gastric carcinoma cell lines and mechanisms of drug resistance

    doi: 10.1038/srep11634

    Figure Lengend Snippet: Down-regulation and mutation of the PTEN gene, as well as up-regulated phosphorylation of AKT and IGF-1R proteins, in trastuzumab-resistant NCI-N87/TR cells. a . Western blotting showed the expression of Her-2, PTEN, EGFR, P-EGFR, IGF-1R, P-IGF-1R, AKT, P-AKT, survivin, CDK2, and p27kipl proteins in NCI-N87 cells and NCI-N87/TR cells. Actin or tubulin expression indicated equal loading. All the gels run under the same experimental conditions and the experiments were repeated 3 times. The representative images were cropped and shown. Data were presented as mean ± SD and analyzed using Student’s t-test. b . Expression of the Her-2 and PTEN genes in NCI-N87 cells and NCI-N87/TR cells shown by real-time PCR. The experiments were repeated 3 times. Data were presented as mean ± SD and analyzed using Student’s t-test. c . Results of PTEN exon 5 sequencing. Mutations at 1309 bp and 1392 bp were detected in NCI-N87/TR cells (A→G and G→A), with the former inducing (H→R; H: His, R: Arg) and the latter inducing (A→T; A: Ala, T: Thr). d . Results of PTEN exon7 sequencing. A mutation at 1732 bp was found in NCI-N87 cells (G→A), inducing (R→Q; R: Arg, Q: Gln), while a mutation at 1763 bp (T→A) was silent. e . Results of PTEN exon 8 sequencing. Mutations at 1955 bp and 1968 bp were found in NCI-N87/TR cells (T→C and A→G), with the former being silent and the latter inducing (K→E; K: Lys, E: Glu).

    Article Snippet: Antibodies directed against Her-2, PTEN, EGFR/P-EGFR, IGF-1R/P-IGF-1R, AKT/P-AKT, survivin, cdk2, and p27kipl proteins were obtained from Abcam, while β-actin and α-tubulin antibodies were obtained from Boster and Sigma, respectively.

    Techniques: Mutagenesis, Western Blot, Expressing, Real-time Polymerase Chain Reaction, Sequencing

    Secretion of the immunodominant antigen is not required for immunodominance. (A) Schematic representation of the B8-mCherry fusion proteins; the location of the signal peptide, GGSGGS linker, TMD, and mCherry are depicted. (B) B8TMmC is not secreted. HeLa cells were infected at an MOI of 5 with B8TMmC or B8mC. Cells and supernatant were harvested at 4 hpi for subcellular fractionation and mCherry and EGFR expression was determined by western blot; equal loading and transfer of samples was confirmed with ponceau S red (P-Red) staining. CE = cytoplasmic extract; ME = membrane extract; SN = supernatant. Data are representative of two independent experiments. (C, D) Comparable CTL priming by B8TMmC and B8mC. CD8 + T cell responses in the spleen of B6 (n = 5) i.n. infected with 5 x 10 3 pfu (C) and 1.5 x 10 4 pfu (D) B8TMmC or B8mC were determined by ex vivo restimulation with CPXV peptides and ICS at 8 dpi. Data are representative of two independent experiments. (E) Cell-associated antigen is cross-presented more efficiently than soluble antigen. B8-specific CD8 + T cell responses in the spleen of B6 (n = 5) i.n. infected with 1.5 x 10 5 pfu B8TMmC or B8mC were determined by tetramer staining at 8, 9, and 10 dpi. Data are representative of two independent experiments. (F) CD8 + T cell responses require BATF3 + DCs. B6 and Batf3 -/- mice (n = 7–10) were i.n. infected with 5 x 10 3 pfu B8TMmC or B8mC and the B8-specific CD8 + T cell responses in the spleen were determined at 6 dpi. n = 3 mock-infected mice. Data are the combined results of three independent experiments.

    Journal: PLoS Pathogens

    Article Title: Cross-priming induces immunodomination in the presence of viral MHC class I inhibition

    doi: 10.1371/journal.ppat.1006883

    Figure Lengend Snippet: Secretion of the immunodominant antigen is not required for immunodominance. (A) Schematic representation of the B8-mCherry fusion proteins; the location of the signal peptide, GGSGGS linker, TMD, and mCherry are depicted. (B) B8TMmC is not secreted. HeLa cells were infected at an MOI of 5 with B8TMmC or B8mC. Cells and supernatant were harvested at 4 hpi for subcellular fractionation and mCherry and EGFR expression was determined by western blot; equal loading and transfer of samples was confirmed with ponceau S red (P-Red) staining. CE = cytoplasmic extract; ME = membrane extract; SN = supernatant. Data are representative of two independent experiments. (C, D) Comparable CTL priming by B8TMmC and B8mC. CD8 + T cell responses in the spleen of B6 (n = 5) i.n. infected with 5 x 10 3 pfu (C) and 1.5 x 10 4 pfu (D) B8TMmC or B8mC were determined by ex vivo restimulation with CPXV peptides and ICS at 8 dpi. Data are representative of two independent experiments. (E) Cell-associated antigen is cross-presented more efficiently than soluble antigen. B8-specific CD8 + T cell responses in the spleen of B6 (n = 5) i.n. infected with 1.5 x 10 5 pfu B8TMmC or B8mC were determined by tetramer staining at 8, 9, and 10 dpi. Data are representative of two independent experiments. (F) CD8 + T cell responses require BATF3 + DCs. B6 and Batf3 -/- mice (n = 7–10) were i.n. infected with 5 x 10 3 pfu B8TMmC or B8mC and the B8-specific CD8 + T cell responses in the spleen were determined at 6 dpi. n = 3 mock-infected mice. Data are the combined results of three independent experiments.

    Article Snippet: Immunoblotting was performed using rabbit polyclonal anti-mCherry and rabbit monoclonal anti-EGFR (Abcam) followed by horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG (Cell Signalling).

    Techniques: Infection, Fractionation, Expressing, Western Blot, Staining, CTL Assay, Ex Vivo, Mouse Assay