p her2 erbb2  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc p her2 erbb2
    ( A, B ) Control and <t>ErbB2-overexpressing</t> MCF-7 cells (high levels) were treated with CDK4i or DMSO 12 h after HRG stimulation and then harvested at 24 h post-HRG stimulation. Western blotting results (A) showing p-ErbB2, p-RB (Ser 807/811 ), p-RB (Thr 373 ), RB, p-AKT (Thr 308 ), AKT, p-ERK (Thr 202 /Thr 204 ), ERK, c-Myc, cyclin D1, and p27 expression (in control cells, i.e., DMSO treatment) after normalization with GAPDH (B). ( C ) Control and ErbB2-overexpressing cells (moderate and high levels) were individually treated with 250 nM CDK4i or DMSO (no treatment) 12 h after stimulation with 10 nM HRG, and images were acquired every 20 min until 72 h after HRG stimulation. Scale bar: 100 µm. ( D ) Images in (C) were analyzed to quantitatively determine the proportion (%) of each cell-cycle phase in the entire set of images; n = 3. ( E ) Cells that completed the M/G1 transition within 72 h out of the n = 100 individually tracked cells were used. Control cells; CDK4i n = 92, DMSO n = 94, ErbB2 OE-Moderate cells; CDK4i n = 53, DMSO n = 88, ErbB2 OE-High; CDK4i n = 81, DMSO n = 91, with the time to G1/S transition plotted on the x-axis and the time to M/G1 transition plotted on the y-axis. The slope (s) and intercept (i) obtained from the regression equation are shown. The red and blue colors indicate the CDK4i and DMSO treatments, respectively. The mean ± SE time taken to reach the respective time to G1/S transition is shown at the bottom of the graph. ( F ) Using the same data as in E, the time to reach the G1/S transition (G1), the duration of the S/G2/M phase (SG2M), and the overall cell-cycle length (G1+SG2M) were assessed for differences in mean time between the CDK4i and DMSO conditions. ( G ) The number of nuclei increased from 16 h to 72 h after HRG stimulation; n = 3). In (B) and (G), * P < 0.05 (Tukey test).
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    1) Product Images from "ErbB2/HER2 expression level determines CDK4-inhibitor sensitivity and cyclin D1 and c-Myc dependency at the G1/S transition"

    Article Title: ErbB2/HER2 expression level determines CDK4-inhibitor sensitivity and cyclin D1 and c-Myc dependency at the G1/S transition

    Journal: bioRxiv

    doi: 10.1101/2024.05.09.593450

    ( A, B ) Control and ErbB2-overexpressing MCF-7 cells (high levels) were treated with CDK4i or DMSO 12 h after HRG stimulation and then harvested at 24 h post-HRG stimulation. Western blotting results (A) showing p-ErbB2, p-RB (Ser 807/811 ), p-RB (Thr 373 ), RB, p-AKT (Thr 308 ), AKT, p-ERK (Thr 202 /Thr 204 ), ERK, c-Myc, cyclin D1, and p27 expression (in control cells, i.e., DMSO treatment) after normalization with GAPDH (B). ( C ) Control and ErbB2-overexpressing cells (moderate and high levels) were individually treated with 250 nM CDK4i or DMSO (no treatment) 12 h after stimulation with 10 nM HRG, and images were acquired every 20 min until 72 h after HRG stimulation. Scale bar: 100 µm. ( D ) Images in (C) were analyzed to quantitatively determine the proportion (%) of each cell-cycle phase in the entire set of images; n = 3. ( E ) Cells that completed the M/G1 transition within 72 h out of the n = 100 individually tracked cells were used. Control cells; CDK4i n = 92, DMSO n = 94, ErbB2 OE-Moderate cells; CDK4i n = 53, DMSO n = 88, ErbB2 OE-High; CDK4i n = 81, DMSO n = 91, with the time to G1/S transition plotted on the x-axis and the time to M/G1 transition plotted on the y-axis. The slope (s) and intercept (i) obtained from the regression equation are shown. The red and blue colors indicate the CDK4i and DMSO treatments, respectively. The mean ± SE time taken to reach the respective time to G1/S transition is shown at the bottom of the graph. ( F ) Using the same data as in E, the time to reach the G1/S transition (G1), the duration of the S/G2/M phase (SG2M), and the overall cell-cycle length (G1+SG2M) were assessed for differences in mean time between the CDK4i and DMSO conditions. ( G ) The number of nuclei increased from 16 h to 72 h after HRG stimulation; n = 3). In (B) and (G), * P < 0.05 (Tukey test).
    Figure Legend Snippet: ( A, B ) Control and ErbB2-overexpressing MCF-7 cells (high levels) were treated with CDK4i or DMSO 12 h after HRG stimulation and then harvested at 24 h post-HRG stimulation. Western blotting results (A) showing p-ErbB2, p-RB (Ser 807/811 ), p-RB (Thr 373 ), RB, p-AKT (Thr 308 ), AKT, p-ERK (Thr 202 /Thr 204 ), ERK, c-Myc, cyclin D1, and p27 expression (in control cells, i.e., DMSO treatment) after normalization with GAPDH (B). ( C ) Control and ErbB2-overexpressing cells (moderate and high levels) were individually treated with 250 nM CDK4i or DMSO (no treatment) 12 h after stimulation with 10 nM HRG, and images were acquired every 20 min until 72 h after HRG stimulation. Scale bar: 100 µm. ( D ) Images in (C) were analyzed to quantitatively determine the proportion (%) of each cell-cycle phase in the entire set of images; n = 3. ( E ) Cells that completed the M/G1 transition within 72 h out of the n = 100 individually tracked cells were used. Control cells; CDK4i n = 92, DMSO n = 94, ErbB2 OE-Moderate cells; CDK4i n = 53, DMSO n = 88, ErbB2 OE-High; CDK4i n = 81, DMSO n = 91, with the time to G1/S transition plotted on the x-axis and the time to M/G1 transition plotted on the y-axis. The slope (s) and intercept (i) obtained from the regression equation are shown. The red and blue colors indicate the CDK4i and DMSO treatments, respectively. The mean ± SE time taken to reach the respective time to G1/S transition is shown at the bottom of the graph. ( F ) Using the same data as in E, the time to reach the G1/S transition (G1), the duration of the S/G2/M phase (SG2M), and the overall cell-cycle length (G1+SG2M) were assessed for differences in mean time between the CDK4i and DMSO conditions. ( G ) The number of nuclei increased from 16 h to 72 h after HRG stimulation; n = 3). In (B) and (G), * P < 0.05 (Tukey test).

    Techniques Used: Western Blot, Expressing

    ( A–G ) ( A - G ) MCF-7 cells overexpressing ErbB2 (high levels) were used. ( A, B ) The following conditions were used: treatment with 250 nM CDK4i 12 h after HRG stimulation, followed by wash/no-wash treatment 4 h later; cells were harvested 40 h after HRG stimulation. Western blotting results showing the expression of p-RB, c-Myc, cyclin D1, p27, and GAPDH. After individually normalizing the proteins relative to GAPDH, the ratio to the no-wash condition was quantified; n = 3. ( C ) Washing operations were performed under the same conditions as in (A, B); cells were fixed 40 h after HRG stimulation and immunostained with p-c-Myc and cyclin D1 antibodies together with DAPI; n = 3,859. The x-axis shows the fluorescence intensity of DAPI, and the y-axis shows the p-c-Myc/cyclin D1 ratio. A higher value on the y-axis indicates that the amount of p-c-Myc in one cell was greater than that of cyclin D1. The black circle shows the vertex of each histogram, and the red dotted line indicates the G1/S transition time point. ( D, E ) Cells were treated for 30 min with/without 64 nM c-Myc inhibitor (EN4) 11.5 h after HRG stimulation. The cells were then incubated with 250 nM CDK4i for 8 h, washed, and imaged 52 h later (72 h after HRG stimulation; D). ( E ) The percentage distributions of G1 and S/G2/M phases were examined based on the images in (D); n = 3. Scale bar: 100 µm. ( F ) Twelve hours after HRG stimulation, the cells were treated with 250 nM CDK4i or DMSO (control). mRNA levels of p27 up to 20 h after HRG stimulation were examined by qPCR. ( G ) After the cells were treated with or without EN4 under the same conditions as in (D–E), RNA was collected 8 h after treatment with inhibitors, and the mRNA levels of CDKN1B were examined by qPCR; n = 3. In (B), (E), (G), * P < 0.05 ( t -test).
    Figure Legend Snippet: ( A–G ) ( A - G ) MCF-7 cells overexpressing ErbB2 (high levels) were used. ( A, B ) The following conditions were used: treatment with 250 nM CDK4i 12 h after HRG stimulation, followed by wash/no-wash treatment 4 h later; cells were harvested 40 h after HRG stimulation. Western blotting results showing the expression of p-RB, c-Myc, cyclin D1, p27, and GAPDH. After individually normalizing the proteins relative to GAPDH, the ratio to the no-wash condition was quantified; n = 3. ( C ) Washing operations were performed under the same conditions as in (A, B); cells were fixed 40 h after HRG stimulation and immunostained with p-c-Myc and cyclin D1 antibodies together with DAPI; n = 3,859. The x-axis shows the fluorescence intensity of DAPI, and the y-axis shows the p-c-Myc/cyclin D1 ratio. A higher value on the y-axis indicates that the amount of p-c-Myc in one cell was greater than that of cyclin D1. The black circle shows the vertex of each histogram, and the red dotted line indicates the G1/S transition time point. ( D, E ) Cells were treated for 30 min with/without 64 nM c-Myc inhibitor (EN4) 11.5 h after HRG stimulation. The cells were then incubated with 250 nM CDK4i for 8 h, washed, and imaged 52 h later (72 h after HRG stimulation; D). ( E ) The percentage distributions of G1 and S/G2/M phases were examined based on the images in (D); n = 3. Scale bar: 100 µm. ( F ) Twelve hours after HRG stimulation, the cells were treated with 250 nM CDK4i or DMSO (control). mRNA levels of p27 up to 20 h after HRG stimulation were examined by qPCR. ( G ) After the cells were treated with or without EN4 under the same conditions as in (D–E), RNA was collected 8 h after treatment with inhibitors, and the mRNA levels of CDKN1B were examined by qPCR; n = 3. In (B), (E), (G), * P < 0.05 ( t -test).

    Techniques Used: Western Blot, Expressing, Fluorescence, Incubation

    (A) In WT MCF-7 cells, the G1/S transition is facilitated by the regulation of cyclin D1/CDK4 via ERK and AKT activities. Conversely, CDK4 inhibition leads to reduced signaling activity of AKT and ERK due to ErbB2 degradation via Hsp90 dis-colocalization, leading to attenuated transcriptional activity of c-Myc activated by epigenetic changes. Consequently, a subpopulation of cells that is unresponsive to CDK4is exhibits a prolonged G1/S transition time. ( B ) In MCF-7 cells expressing high levels of high ErbB2, increased AKT activity leads to increased c-Myc expression. Subsequently, the expression of p27, which is critical for the stability of cyclin D1/CDK4 during the G1 phase, is suppressed by c-Myc, resulting in a slightly delayed G1/S entry compared to that in wild-type cells. Conversely, following CDK4 inhibition, most cells exhibit a cell-cycle arrest. This arrest is reversibly maintained by the strong transcriptional activity of c-Myc, ultimately sustaining survival signals.
    Figure Legend Snippet: (A) In WT MCF-7 cells, the G1/S transition is facilitated by the regulation of cyclin D1/CDK4 via ERK and AKT activities. Conversely, CDK4 inhibition leads to reduced signaling activity of AKT and ERK due to ErbB2 degradation via Hsp90 dis-colocalization, leading to attenuated transcriptional activity of c-Myc activated by epigenetic changes. Consequently, a subpopulation of cells that is unresponsive to CDK4is exhibits a prolonged G1/S transition time. ( B ) In MCF-7 cells expressing high levels of high ErbB2, increased AKT activity leads to increased c-Myc expression. Subsequently, the expression of p27, which is critical for the stability of cyclin D1/CDK4 during the G1 phase, is suppressed by c-Myc, resulting in a slightly delayed G1/S entry compared to that in wild-type cells. Conversely, following CDK4 inhibition, most cells exhibit a cell-cycle arrest. This arrest is reversibly maintained by the strong transcriptional activity of c-Myc, ultimately sustaining survival signals.

    Techniques Used: Inhibition, Activity Assay, Expressing

    p her2 erbb2 tyr877  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc p her2 erbb2 tyr877
    P Her2 Erbb2 Tyr877, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    p her2 erbb2 tyr877  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc p her2 erbb2 tyr877
    P Her2 Erbb2 Tyr877, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    p-her2/erbb2 y122  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc p-her2/erbb2 y122
    P Her2/Erbb2 Y122, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 96/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    p her2 erbb2  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc p her2 erbb2
    ERBB signaling constitutes the core of KRAS dependency (A) PPI network of the 30 genes generated with STRING and visualized with Cytoscape. Colors indicate log 2 FC (nodes’ size indicates absolute log 2 FC) in gene expression between 20 most mt KRAS-dependent and 20 most mt KRAS-independent cell lines. (B) Results from GO enrichment analysis of the 30 genes using ShinyGO. Only terms with FDR<0.01 are plotted. Expression of genes from each category was averaged among cell lines with smallest and highest KDS30 scores. ‘Any (L)’ represents mt KRAS cancer cell lines with 10 lowest KDS30 scores. ‘Any (H)’ represents mt KRAS cancer cell lines with 10 highest KDS30 scores. ‘Panc (L)’ and ‘Panc (H)’ represent mt KRAS pancreatic cancer cell lines with 4 lowest and 4 highest KDS30 scores, respectively. ‘Lung (L)’ and ‘Lung (H)’ represent mt KRAS lung cancer cell lines with 4 lowest and 4 highest KDS30 scores, respectively. (C and E) <t>EGFR/ERBB2</t> (in blue), MEK (in red) and RAF (in green) inhibitors that were found to selectively kill high KDS30 over low KDS30 PAAD (in C) or LUAD (in E) cancer cells, considering max top 40 drugs. PRISM_1ry: primary PRISM Repurposing screen, PRISM_2ry: secondary PRISM Repurposing screen, CTRP: Cancer Therapeutics Response Portal v2. Y-axis: difference in mean FC or mean AUC, see main text. Only inhibitors of EGFR/ERBB2/MEK/RAF with p<0.05 are shown. (D and F) Results from drug sensitivity data analysis for mt KRAS pancreatic cancer cells included in the PRISM 2ry screen (D) and mt KRAS lung cancer cell lines included in the CTRP screen (F). Mean AUC of high KDS30 cells was subtracted from mean AUC of low KDS30 cells and plotted on X-axis. See main text for thresholds. Student’s t - test was applied to determine the statistical significance to observe the difference and plotted on Y-axis. MEK inhibitors are highlighted in red. EGFR/ERBB2 inhibitors are highlighted in blue. NERA: neratinib; SELU: selumetinib; TRAM: trametinib; PD31: PD-318088; AFAT: afatinib; GEFI: gefitinib; AZD8: AZD8931; DACO: dacomitinib; TUCA: tucatinib; CANE: canertinib; COBI: cobimetinib; AS70: AS-703026; OSIM: Osimertinib; ERLO: erlotinib . See also <xref ref-type=Figure S3 . " width="250" height="auto" />
    P Her2 Erbb2, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 96/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "Novel mutant KRAS addiction signature predicts response to the combination of ERBB and MEK inhibitors in lung and pancreatic cancers"

    Article Title: Novel mutant KRAS addiction signature predicts response to the combination of ERBB and MEK inhibitors in lung and pancreatic cancers

    Journal: iScience

    doi: 10.1016/j.isci.2023.106082

    ERBB signaling constitutes the core of KRAS dependency (A) PPI network of the 30 genes generated with STRING and visualized with Cytoscape. Colors indicate log 2 FC (nodes’ size indicates absolute log 2 FC) in gene expression between 20 most mt KRAS-dependent and 20 most mt KRAS-independent cell lines. (B) Results from GO enrichment analysis of the 30 genes using ShinyGO. Only terms with FDR<0.01 are plotted. Expression of genes from each category was averaged among cell lines with smallest and highest KDS30 scores. ‘Any (L)’ represents mt KRAS cancer cell lines with 10 lowest KDS30 scores. ‘Any (H)’ represents mt KRAS cancer cell lines with 10 highest KDS30 scores. ‘Panc (L)’ and ‘Panc (H)’ represent mt KRAS pancreatic cancer cell lines with 4 lowest and 4 highest KDS30 scores, respectively. ‘Lung (L)’ and ‘Lung (H)’ represent mt KRAS lung cancer cell lines with 4 lowest and 4 highest KDS30 scores, respectively. (C and E) EGFR/ERBB2 (in blue), MEK (in red) and RAF (in green) inhibitors that were found to selectively kill high KDS30 over low KDS30 PAAD (in C) or LUAD (in E) cancer cells, considering max top 40 drugs. PRISM_1ry: primary PRISM Repurposing screen, PRISM_2ry: secondary PRISM Repurposing screen, CTRP: Cancer Therapeutics Response Portal v2. Y-axis: difference in mean FC or mean AUC, see main text. Only inhibitors of EGFR/ERBB2/MEK/RAF with p<0.05 are shown. (D and F) Results from drug sensitivity data analysis for mt KRAS pancreatic cancer cells included in the PRISM 2ry screen (D) and mt KRAS lung cancer cell lines included in the CTRP screen (F). Mean AUC of high KDS30 cells was subtracted from mean AUC of low KDS30 cells and plotted on X-axis. See main text for thresholds. Student’s t - test was applied to determine the statistical significance to observe the difference and plotted on Y-axis. MEK inhibitors are highlighted in red. EGFR/ERBB2 inhibitors are highlighted in blue. NERA: neratinib; SELU: selumetinib; TRAM: trametinib; PD31: PD-318088; AFAT: afatinib; GEFI: gefitinib; AZD8: AZD8931; DACO: dacomitinib; TUCA: tucatinib; CANE: canertinib; COBI: cobimetinib; AS70: AS-703026; OSIM: Osimertinib; ERLO: erlotinib . See also <xref ref-type=Figure S3 . " title="... 4 highest KDS30 scores, respectively. (C and E) EGFR/ERBB2 (in blue), MEK (in red) and RAF (in ..." property="contentUrl" width="100%" height="100%"/>
    Figure Legend Snippet: ERBB signaling constitutes the core of KRAS dependency (A) PPI network of the 30 genes generated with STRING and visualized with Cytoscape. Colors indicate log 2 FC (nodes’ size indicates absolute log 2 FC) in gene expression between 20 most mt KRAS-dependent and 20 most mt KRAS-independent cell lines. (B) Results from GO enrichment analysis of the 30 genes using ShinyGO. Only terms with FDR<0.01 are plotted. Expression of genes from each category was averaged among cell lines with smallest and highest KDS30 scores. ‘Any (L)’ represents mt KRAS cancer cell lines with 10 lowest KDS30 scores. ‘Any (H)’ represents mt KRAS cancer cell lines with 10 highest KDS30 scores. ‘Panc (L)’ and ‘Panc (H)’ represent mt KRAS pancreatic cancer cell lines with 4 lowest and 4 highest KDS30 scores, respectively. ‘Lung (L)’ and ‘Lung (H)’ represent mt KRAS lung cancer cell lines with 4 lowest and 4 highest KDS30 scores, respectively. (C and E) EGFR/ERBB2 (in blue), MEK (in red) and RAF (in green) inhibitors that were found to selectively kill high KDS30 over low KDS30 PAAD (in C) or LUAD (in E) cancer cells, considering max top 40 drugs. PRISM_1ry: primary PRISM Repurposing screen, PRISM_2ry: secondary PRISM Repurposing screen, CTRP: Cancer Therapeutics Response Portal v2. Y-axis: difference in mean FC or mean AUC, see main text. Only inhibitors of EGFR/ERBB2/MEK/RAF with p<0.05 are shown. (D and F) Results from drug sensitivity data analysis for mt KRAS pancreatic cancer cells included in the PRISM 2ry screen (D) and mt KRAS lung cancer cell lines included in the CTRP screen (F). Mean AUC of high KDS30 cells was subtracted from mean AUC of low KDS30 cells and plotted on X-axis. See main text for thresholds. Student’s t - test was applied to determine the statistical significance to observe the difference and plotted on Y-axis. MEK inhibitors are highlighted in red. EGFR/ERBB2 inhibitors are highlighted in blue. NERA: neratinib; SELU: selumetinib; TRAM: trametinib; PD31: PD-318088; AFAT: afatinib; GEFI: gefitinib; AZD8: AZD8931; DACO: dacomitinib; TUCA: tucatinib; CANE: canertinib; COBI: cobimetinib; AS70: AS-703026; OSIM: Osimertinib; ERLO: erlotinib . See also Figure S3 .

    Techniques Used: Generated, Expressing

    mt KRAS addicted lung cancer lines are highly sensitive to the combination of EGFR/ERBB2 inhibitors with MEK inhibitors High KDS30 H358 and low KDS30 LU99 lung cancer cells were treated with vehicle control (Ctrl), an EGFR/ERBB2 inhibitor and a MEK inhibitor, either alone or in combination as indicated on each figure panel, for 48 h and processed for western blotting as described in . pEGFR, pErbB2, pERK: phosphorylated EGFR, ErbB2, and ERK, respectively. c-Casp3 and c-PARP: cleaved Casp3 and PARP, respectively. Actin: loading control. Single ∗ and double ∗∗ indicate gels each protein is blotted from. See also <xref ref-type=Figure S4 . " title="... lines are highly sensitive to the combination of EGFR/ERBB2 inhibitors with MEK inhibitors High KDS30 H358 and ..." property="contentUrl" width="100%" height="100%"/>
    Figure Legend Snippet: mt KRAS addicted lung cancer lines are highly sensitive to the combination of EGFR/ERBB2 inhibitors with MEK inhibitors High KDS30 H358 and low KDS30 LU99 lung cancer cells were treated with vehicle control (Ctrl), an EGFR/ERBB2 inhibitor and a MEK inhibitor, either alone or in combination as indicated on each figure panel, for 48 h and processed for western blotting as described in . pEGFR, pErbB2, pERK: phosphorylated EGFR, ErbB2, and ERK, respectively. c-Casp3 and c-PARP: cleaved Casp3 and PARP, respectively. Actin: loading control. Single ∗ and double ∗∗ indicate gels each protein is blotted from. See also Figure S4 .

    Techniques Used: Western Blot


    Figure Legend Snippet:

    Techniques Used: Recombinant, Expressing, CRISPR, Software

    p her2  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc p her2
    Clinicopathological characteristics of breast cancer patients.
    P Her2, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "Tumorigenicity of EGFR- and/or HER2-Positive Breast Cancers Is Mediated by Recruitment of Tumor-Associated Macrophages"

    Article Title: Tumorigenicity of EGFR- and/or HER2-Positive Breast Cancers Is Mediated by Recruitment of Tumor-Associated Macrophages

    Journal: International Journal of Molecular Sciences

    doi: 10.3390/ijms24021443

    Clinicopathological characteristics of breast cancer patients.
    Figure Legend Snippet: Clinicopathological characteristics of breast cancer patients.

    Techniques Used:

    Co-expression of EGFR and HER2 correlates with poor survival in breast cancer patients. ( A ) Disease-free survival rates were compared between EGFR+/HER2-/ER- ( n = 92) and EGFR+/HER2+/ER- ( n = 23) patients ( p = 0.0229). ( B ) Overall survival rates were compared between EGFR+/HER2-/ER- ( n = 92) and EGFR+/HER2+/ER- ( n = 23) patients ( p = 0.019).
    Figure Legend Snippet: Co-expression of EGFR and HER2 correlates with poor survival in breast cancer patients. ( A ) Disease-free survival rates were compared between EGFR+/HER2-/ER- ( n = 92) and EGFR+/HER2+/ER- ( n = 23) patients ( p = 0.0229). ( B ) Overall survival rates were compared between EGFR+/HER2-/ER- ( n = 92) and EGFR+/HER2+/ER- ( n = 23) patients ( p = 0.019).

    Techniques Used: Expressing

    Co-expression of EGFR and HER2 promotes invasion and proliferation. ( A ) Transcript levels of EGFR and HER2 in Vec and HER2-overexpressed MDA-MB231 cells were analyzed using quantitative reverse transcription polymerase chain reaction (RT-qPCR). Values were normalized to ACTB . ( B ) Levels of protein expression were analyzed using Western blotting with indicated antibodies. β-actin was used as a loading control. ( C ) Invasion assays were performed using MDA-MB231 Vec and HER2-overexpressed cells as described in the method section. The scale bar represents 200 μm. ( D ) Cell cycle analysis was performed using MDA-MB231 Vec and HER2-overexpressed cells. * p < 0.05.
    Figure Legend Snippet: Co-expression of EGFR and HER2 promotes invasion and proliferation. ( A ) Transcript levels of EGFR and HER2 in Vec and HER2-overexpressed MDA-MB231 cells were analyzed using quantitative reverse transcription polymerase chain reaction (RT-qPCR). Values were normalized to ACTB . ( B ) Levels of protein expression were analyzed using Western blotting with indicated antibodies. β-actin was used as a loading control. ( C ) Invasion assays were performed using MDA-MB231 Vec and HER2-overexpressed cells as described in the method section. The scale bar represents 200 μm. ( D ) Cell cycle analysis was performed using MDA-MB231 Vec and HER2-overexpressed cells. * p < 0.05.

    Techniques Used: Expressing, Reverse Transcription Polymerase Chain Reaction, Quantitative RT-PCR, Western Blot, Cell Cycle Assay

    Cytokine secretion profile is altered by HER2 overexpression. ( A ) Human cytokine array was performed using conditioned culture media of MDA-MB231 Vec and HER2-overexpressed cells. ( B – E ) Transcript levels of CCL2 , CCL5 , IL-6 , and IL-8 in Vec and HER2-overexpressed MDA-MB231 cells were analyzed using RT-qPCR. Values were normalized to ACTB . * p < 0.05.
    Figure Legend Snippet: Cytokine secretion profile is altered by HER2 overexpression. ( A ) Human cytokine array was performed using conditioned culture media of MDA-MB231 Vec and HER2-overexpressed cells. ( B – E ) Transcript levels of CCL2 , CCL5 , IL-6 , and IL-8 in Vec and HER2-overexpressed MDA-MB231 cells were analyzed using RT-qPCR. Values were normalized to ACTB . * p < 0.05.

    Techniques Used: Over Expression, Quantitative RT-PCR

    CCL2 expression is downregulated by neratinib. ( A , B ) Each cell line was treated with 5 μM neratinib for 24 h. ( A ) Transcript levels of CCL2 were analyzed using RT-qPCR. Values were normalized to ACTB . ( B ) ELISA was performed using conditioned culture media of Vec and HER2-overexpressed MDA-MB231 cells. ( C ) Levels of protein expression were analyzed using Western blotting with indicated antibodies. β-actin was used as a loading control. ( D ) ELISA was performed using conditioned culture media of MDA453 and BT474 cells transfected with scrambled or HER2-specific siRNA. * p < 0.05; ** p < 0.01; ϕ p < 0.05.
    Figure Legend Snippet: CCL2 expression is downregulated by neratinib. ( A , B ) Each cell line was treated with 5 μM neratinib for 24 h. ( A ) Transcript levels of CCL2 were analyzed using RT-qPCR. Values were normalized to ACTB . ( B ) ELISA was performed using conditioned culture media of Vec and HER2-overexpressed MDA-MB231 cells. ( C ) Levels of protein expression were analyzed using Western blotting with indicated antibodies. β-actin was used as a loading control. ( D ) ELISA was performed using conditioned culture media of MDA453 and BT474 cells transfected with scrambled or HER2-specific siRNA. * p < 0.05; ** p < 0.01; ϕ p < 0.05.

    Techniques Used: Expressing, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Western Blot, Transfection

    CCL2 mediates motility of TAMs. ( A ) Immunofluorescence confocal microscopy was performed using M0 and M2-differentiated THP-1 cells. Anti-CD11b primary antibody and Alexa Fluor 488-conjugated secondary antibody were used for staining. The nucleus was stained with DAPI. ( B ) Upper, levels of CD-11b and CD163 protein expression were analyzed using Western blotting with whole cell lysates from M0 and M2-differentiated THP-1 cells with indicated antibodies. β-actin was used as a loading control. Lower, transcript levels of CD11b in M0 and M2-differentiated THP-1 cells were analyzed using RT-qPCR. Values were normalized to ACTB. ( C ) Migration assays were performed as described in the method section. ( D ) Cytokine arrays were performed using whole cell lysates of control and CCL2-treated M2-differentiated THP-1 cells. ( E ) Migration assays were performed with M2-differentiated THP-1 cells treated with conditioned culture media from MDA-MB231 Vec and HER2-overexpressed cells. The scale bar represents 200 μm. ( F ) RT-qPCR showing transcript levels of IL-8 and IL-1B in M2-differentiated THP-1 cells treated with conditioned culture media from MDA-MB231 Vec and HER2-overexpressed cells. Values were normalized to ACTB . * p < 0.05.
    Figure Legend Snippet: CCL2 mediates motility of TAMs. ( A ) Immunofluorescence confocal microscopy was performed using M0 and M2-differentiated THP-1 cells. Anti-CD11b primary antibody and Alexa Fluor 488-conjugated secondary antibody were used for staining. The nucleus was stained with DAPI. ( B ) Upper, levels of CD-11b and CD163 protein expression were analyzed using Western blotting with whole cell lysates from M0 and M2-differentiated THP-1 cells with indicated antibodies. β-actin was used as a loading control. Lower, transcript levels of CD11b in M0 and M2-differentiated THP-1 cells were analyzed using RT-qPCR. Values were normalized to ACTB. ( C ) Migration assays were performed as described in the method section. ( D ) Cytokine arrays were performed using whole cell lysates of control and CCL2-treated M2-differentiated THP-1 cells. ( E ) Migration assays were performed with M2-differentiated THP-1 cells treated with conditioned culture media from MDA-MB231 Vec and HER2-overexpressed cells. The scale bar represents 200 μm. ( F ) RT-qPCR showing transcript levels of IL-8 and IL-1B in M2-differentiated THP-1 cells treated with conditioned culture media from MDA-MB231 Vec and HER2-overexpressed cells. Values were normalized to ACTB . * p < 0.05.

    Techniques Used: Immunofluorescence, Confocal Microscopy, Staining, Expressing, Western Blot, Quantitative RT-PCR, Migration

    TAMs recruited into HER2-overexpressed tumors. ( A ) A schematic diagram illustrating the xenograft study in this figure: MDA-MB231 Vec or HER2-overexpressed cells were injected into the 2nd fat pad of NOD/SCID mice. Recruited TAMs were monitored after 12 weeks. ( B ) Recruited TAMs were analyzed using CD163 staining.
    Figure Legend Snippet: TAMs recruited into HER2-overexpressed tumors. ( A ) A schematic diagram illustrating the xenograft study in this figure: MDA-MB231 Vec or HER2-overexpressed cells were injected into the 2nd fat pad of NOD/SCID mice. Recruited TAMs were monitored after 12 weeks. ( B ) Recruited TAMs were analyzed using CD163 staining.

    Techniques Used: Injection, Staining

    p erbb2 tyr1248  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc p erbb2 tyr1248
    Primer sequences used for the qPCR analysis
    P Erbb2 Tyr1248, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "Dysregulation of Neuregulin-1/ErbB signaling in the hippocampus of rats after administration of doxorubicin"

    Article Title: Dysregulation of Neuregulin-1/ErbB signaling in the hippocampus of rats after administration of doxorubicin

    Journal: Drug Design, Development and Therapy

    doi: 10.2147/DDDT.S151511

    Primer sequences used for the qPCR analysis
    Figure Legend Snippet: Primer sequences used for the qPCR analysis

    Techniques Used: Amplification

    Effect of Dox on gene expression of ErbB2, ErbB4, and the ratio of pErbB4/ErbB4 and pErbB2/ErbB2 in the hippocampus. Notes: ErbB4 mRNA expression ( A ); pErbB4/ErbB4 ratio ( B ); ErbB2 mRNA expression ( C ); and pErbB2/ErbB2 ratio ( D ). Data are expressed as mean ± SEM (n=6–7). * p <0.05 and ** p <0.01 compared to the control group. Abbreviations: Dox, doxorubicin; DoxS, doxorubicin administration for short time; DoxL, doxorubicin administration for long time; SEM, standard error of the mean.
    Figure Legend Snippet: Effect of Dox on gene expression of ErbB2, ErbB4, and the ratio of pErbB4/ErbB4 and pErbB2/ErbB2 in the hippocampus. Notes: ErbB4 mRNA expression ( A ); pErbB4/ErbB4 ratio ( B ); ErbB2 mRNA expression ( C ); and pErbB2/ErbB2 ratio ( D ). Data are expressed as mean ± SEM (n=6–7). * p <0.05 and ** p <0.01 compared to the control group. Abbreviations: Dox, doxorubicin; DoxS, doxorubicin administration for short time; DoxL, doxorubicin administration for long time; SEM, standard error of the mean.

    Techniques Used: Expressing

    p her2 erbb2 antibody  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc p her2 erbb2 antibody
    Panel a) is a representative Western blot showing the levels of phosphorylated <t>ErbB2</t> (p-ErbB2) at the indicated tyrosines Y1221/1222, Y1248 (detected by separate antibodies labeled as Y1248 a and <t>Y1248b,</t> Y877, total (t-) ErbB2 and β-actin in the isolated mesenteric bed from normal controls (C), diabetic (D) and diabetic animals treated for 4 weeks with AG825 (1 mg/kg/ alt-diem ; +AG825). Panels b-e) are densitometry histograms showing levels of phosphorylated EGFR at the stated tyrosine residue and panel f) t-ErbB2 normalized to actin whereas panel g) shows the ratio of p-ErbB2 (Y1221/1222) to t-ErbB2. N = 5; Mean±SD. Asterisk (*) indicates significantly different (p<0.05) mean values from normal non-diabetic rats (C) whereas hash (#) indicates significantly different mean values (p<0.05) from diabetic rats (D).
    P Her2 Erbb2 Antibody, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/p her2 erbb2 antibody/product/Cell Signaling Technology Inc
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    1) Product Images from "Activation of ErbB2 and Downstream Signalling via Rho Kinases and ERK1/2 Contributes to Diabetes-Induced Vascular Dysfunction"

    Article Title: Activation of ErbB2 and Downstream Signalling via Rho Kinases and ERK1/2 Contributes to Diabetes-Induced Vascular Dysfunction

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0067813

    Panel a) is a representative Western blot showing the levels of phosphorylated ErbB2 (p-ErbB2) at the indicated tyrosines Y1221/1222, Y1248 (detected by separate antibodies labeled as Y1248 a and Y1248b, Y877, total (t-) ErbB2 and β-actin in the isolated mesenteric bed from normal controls (C), diabetic (D) and diabetic animals treated for 4 weeks with AG825 (1 mg/kg/ alt-diem ; +AG825). Panels b-e) are densitometry histograms showing levels of phosphorylated EGFR at the stated tyrosine residue and panel f) t-ErbB2 normalized to actin whereas panel g) shows the ratio of p-ErbB2 (Y1221/1222) to t-ErbB2. N = 5; Mean±SD. Asterisk (*) indicates significantly different (p<0.05) mean values from normal non-diabetic rats (C) whereas hash (#) indicates significantly different mean values (p<0.05) from diabetic rats (D).
    Figure Legend Snippet: Panel a) is a representative Western blot showing the levels of phosphorylated ErbB2 (p-ErbB2) at the indicated tyrosines Y1221/1222, Y1248 (detected by separate antibodies labeled as Y1248 a and Y1248b, Y877, total (t-) ErbB2 and β-actin in the isolated mesenteric bed from normal controls (C), diabetic (D) and diabetic animals treated for 4 weeks with AG825 (1 mg/kg/ alt-diem ; +AG825). Panels b-e) are densitometry histograms showing levels of phosphorylated EGFR at the stated tyrosine residue and panel f) t-ErbB2 normalized to actin whereas panel g) shows the ratio of p-ErbB2 (Y1221/1222) to t-ErbB2. N = 5; Mean±SD. Asterisk (*) indicates significantly different (p<0.05) mean values from normal non-diabetic rats (C) whereas hash (#) indicates significantly different mean values (p<0.05) from diabetic rats (D).

    Techniques Used: Western Blot, Labeling, Isolation

    Panel a) and c) are represenatative Western Blots following immunoprecipitations (IP) with either total-erbB2 or total-EGFR antibody and subsequent immunoblotting (IB) with both antibodies individually. Panel b) and d) represents the mean ratio of erbB2/EGFR dimers as assessed by densitometry for non-diabetic controls, (C), diabetic (D) and diabetic animals chronically treated with AG825 (+AG825) or AG1478 (+ AG1478) (both at dose of 1 mg/kg/ alt-diem ). N = 4; Asterisk (*) indicates significantly different (p<0.05) mean values from normal non-diabetic rats (C) whereas hash (#) indicates significantly different mean values (p<0.05) from diabetic rats (D).
    Figure Legend Snippet: Panel a) and c) are represenatative Western Blots following immunoprecipitations (IP) with either total-erbB2 or total-EGFR antibody and subsequent immunoblotting (IB) with both antibodies individually. Panel b) and d) represents the mean ratio of erbB2/EGFR dimers as assessed by densitometry for non-diabetic controls, (C), diabetic (D) and diabetic animals chronically treated with AG825 (+AG825) or AG1478 (+ AG1478) (both at dose of 1 mg/kg/ alt-diem ). N = 4; Asterisk (*) indicates significantly different (p<0.05) mean values from normal non-diabetic rats (C) whereas hash (#) indicates significantly different mean values (p<0.05) from diabetic rats (D).

    Techniques Used: Western Blot

    A) Panel i) is a representative Western blot showing total (t-) or phosphorylated (p) levels of the stated proteins in VSMC grown in normal (5.5mM) D-glucose (NG), high glucose (25.5mM D-glucose; HG) or HG cotreated with increasing micromolar doses of AG825 (+ AG825). Panels ii-viii) are densitometry histograms showing total (t-) or phosphorylated (p-) levels of the stated proteins normalized to actin. B) Panel i) is a representative Western blot showing total (t-) or phosphorylated (p) levels of the stated proteins in VSMC grown in high glucose (25.5mM D-glucose; HG) or HG cotreated with increasing doses of anti-ErbB2 siRNA (ErbB2 siRNA) or non-targeting control siRNA (C siRNA). Panels ii-viii) are densitometry histograms showing total (t-) or phosphorylated (p-) levels of the stated proteins normalized to actin. N = 5; Mean±SD. Asterisk (*) indicates significantly different (p<0.05) mean values from normal non-diabetic rats (C) whereas hash (#) indicates significantly different mean values (p<0.05) from diabetic rats (D).
    Figure Legend Snippet: A) Panel i) is a representative Western blot showing total (t-) or phosphorylated (p) levels of the stated proteins in VSMC grown in normal (5.5mM) D-glucose (NG), high glucose (25.5mM D-glucose; HG) or HG cotreated with increasing micromolar doses of AG825 (+ AG825). Panels ii-viii) are densitometry histograms showing total (t-) or phosphorylated (p-) levels of the stated proteins normalized to actin. B) Panel i) is a representative Western blot showing total (t-) or phosphorylated (p) levels of the stated proteins in VSMC grown in high glucose (25.5mM D-glucose; HG) or HG cotreated with increasing doses of anti-ErbB2 siRNA (ErbB2 siRNA) or non-targeting control siRNA (C siRNA). Panels ii-viii) are densitometry histograms showing total (t-) or phosphorylated (p-) levels of the stated proteins normalized to actin. N = 5; Mean±SD. Asterisk (*) indicates significantly different (p<0.05) mean values from normal non-diabetic rats (C) whereas hash (#) indicates significantly different mean values (p<0.05) from diabetic rats (D).

    Techniques Used: Western Blot

    p her2 erbb2 antibody tyr1221 1222  (Cell Signaling Technology Inc)


    Bioz Manufacturer Symbol Cell Signaling Technology Inc manufactures this product  
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    Cell Signaling Technology Inc p her2 erbb2 antibody tyr1221 1222
    Panel a) is a representative Western blot showing the levels of phosphorylated <t>ErbB2</t> (p-ErbB2) at the indicated tyrosines Y1221/1222, Y1248 (detected by separate antibodies labeled as Y1248 a and Y1248b, Y877, total (t-) ErbB2 and β-actin in the isolated mesenteric bed from normal controls (C), diabetic (D) and diabetic animals treated for 4 weeks with AG825 (1 mg/kg/ alt-diem ; +AG825). Panels b-e) are densitometry histograms showing levels of phosphorylated EGFR at the stated tyrosine residue and panel f) t-ErbB2 normalized to actin whereas panel g) shows the ratio of p-ErbB2 (Y1221/1222) to t-ErbB2. N = 5; Mean±SD. Asterisk (*) indicates significantly different (p<0.05) mean values from normal non-diabetic rats (C) whereas hash (#) indicates significantly different mean values (p<0.05) from diabetic rats (D).
    P Her2 Erbb2 Antibody Tyr1221 1222, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 96/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/p her2 erbb2 antibody tyr1221 1222/product/Cell Signaling Technology Inc
    Average 96 stars, based on 1 article reviews
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    Images

    1) Product Images from "Activation of ErbB2 and Downstream Signalling via Rho Kinases and ERK1/2 Contributes to Diabetes-Induced Vascular Dysfunction"

    Article Title: Activation of ErbB2 and Downstream Signalling via Rho Kinases and ERK1/2 Contributes to Diabetes-Induced Vascular Dysfunction

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0067813

    Panel a) is a representative Western blot showing the levels of phosphorylated ErbB2 (p-ErbB2) at the indicated tyrosines Y1221/1222, Y1248 (detected by separate antibodies labeled as Y1248 a and Y1248b, Y877, total (t-) ErbB2 and β-actin in the isolated mesenteric bed from normal controls (C), diabetic (D) and diabetic animals treated for 4 weeks with AG825 (1 mg/kg/ alt-diem ; +AG825). Panels b-e) are densitometry histograms showing levels of phosphorylated EGFR at the stated tyrosine residue and panel f) t-ErbB2 normalized to actin whereas panel g) shows the ratio of p-ErbB2 (Y1221/1222) to t-ErbB2. N = 5; Mean±SD. Asterisk (*) indicates significantly different (p<0.05) mean values from normal non-diabetic rats (C) whereas hash (#) indicates significantly different mean values (p<0.05) from diabetic rats (D).
    Figure Legend Snippet: Panel a) is a representative Western blot showing the levels of phosphorylated ErbB2 (p-ErbB2) at the indicated tyrosines Y1221/1222, Y1248 (detected by separate antibodies labeled as Y1248 a and Y1248b, Y877, total (t-) ErbB2 and β-actin in the isolated mesenteric bed from normal controls (C), diabetic (D) and diabetic animals treated for 4 weeks with AG825 (1 mg/kg/ alt-diem ; +AG825). Panels b-e) are densitometry histograms showing levels of phosphorylated EGFR at the stated tyrosine residue and panel f) t-ErbB2 normalized to actin whereas panel g) shows the ratio of p-ErbB2 (Y1221/1222) to t-ErbB2. N = 5; Mean±SD. Asterisk (*) indicates significantly different (p<0.05) mean values from normal non-diabetic rats (C) whereas hash (#) indicates significantly different mean values (p<0.05) from diabetic rats (D).

    Techniques Used: Western Blot, Labeling, Isolation

    Panel a) and c) are represenatative Western Blots following immunoprecipitations (IP) with either total-erbB2 or total-EGFR antibody and subsequent immunoblotting (IB) with both antibodies individually. Panel b) and d) represents the mean ratio of erbB2/EGFR dimers as assessed by densitometry for non-diabetic controls, (C), diabetic (D) and diabetic animals chronically treated with AG825 (+AG825) or AG1478 (+ AG1478) (both at dose of 1 mg/kg/ alt-diem ). N = 4; Asterisk (*) indicates significantly different (p<0.05) mean values from normal non-diabetic rats (C) whereas hash (#) indicates significantly different mean values (p<0.05) from diabetic rats (D).
    Figure Legend Snippet: Panel a) and c) are represenatative Western Blots following immunoprecipitations (IP) with either total-erbB2 or total-EGFR antibody and subsequent immunoblotting (IB) with both antibodies individually. Panel b) and d) represents the mean ratio of erbB2/EGFR dimers as assessed by densitometry for non-diabetic controls, (C), diabetic (D) and diabetic animals chronically treated with AG825 (+AG825) or AG1478 (+ AG1478) (both at dose of 1 mg/kg/ alt-diem ). N = 4; Asterisk (*) indicates significantly different (p<0.05) mean values from normal non-diabetic rats (C) whereas hash (#) indicates significantly different mean values (p<0.05) from diabetic rats (D).

    Techniques Used: Western Blot

    A) Panel i) is a representative Western blot showing total (t-) or phosphorylated (p) levels of the stated proteins in VSMC grown in normal (5.5mM) D-glucose (NG), high glucose (25.5mM D-glucose; HG) or HG cotreated with increasing micromolar doses of AG825 (+ AG825). Panels ii-viii) are densitometry histograms showing total (t-) or phosphorylated (p-) levels of the stated proteins normalized to actin. B) Panel i) is a representative Western blot showing total (t-) or phosphorylated (p) levels of the stated proteins in VSMC grown in high glucose (25.5mM D-glucose; HG) or HG cotreated with increasing doses of anti-ErbB2 siRNA (ErbB2 siRNA) or non-targeting control siRNA (C siRNA). Panels ii-viii) are densitometry histograms showing total (t-) or phosphorylated (p-) levels of the stated proteins normalized to actin. N = 5; Mean±SD. Asterisk (*) indicates significantly different (p<0.05) mean values from normal non-diabetic rats (C) whereas hash (#) indicates significantly different mean values (p<0.05) from diabetic rats (D).
    Figure Legend Snippet: A) Panel i) is a representative Western blot showing total (t-) or phosphorylated (p) levels of the stated proteins in VSMC grown in normal (5.5mM) D-glucose (NG), high glucose (25.5mM D-glucose; HG) or HG cotreated with increasing micromolar doses of AG825 (+ AG825). Panels ii-viii) are densitometry histograms showing total (t-) or phosphorylated (p-) levels of the stated proteins normalized to actin. B) Panel i) is a representative Western blot showing total (t-) or phosphorylated (p) levels of the stated proteins in VSMC grown in high glucose (25.5mM D-glucose; HG) or HG cotreated with increasing doses of anti-ErbB2 siRNA (ErbB2 siRNA) or non-targeting control siRNA (C siRNA). Panels ii-viii) are densitometry histograms showing total (t-) or phosphorylated (p-) levels of the stated proteins normalized to actin. N = 5; Mean±SD. Asterisk (*) indicates significantly different (p<0.05) mean values from normal non-diabetic rats (C) whereas hash (#) indicates significantly different mean values (p<0.05) from diabetic rats (D).

    Techniques Used: Western Blot

    p her2 erbb2 antibody  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc p her2 erbb2 antibody
    Panel a) is a representative Western blot showing the levels of phosphorylated <t>ErbB2</t> (p-ErbB2) at the indicated tyrosines Y1221/1222, Y1248 (detected by separate antibodies labeled as Y1248 a and <t>Y1248b,</t> Y877, total (t-) ErbB2 and β-actin in the isolated mesenteric bed from normal controls (C), diabetic (D) and diabetic animals treated for 4 weeks with AG825 (1 mg/kg/ alt-diem ; +AG825). Panels b-e) are densitometry histograms showing levels of phosphorylated EGFR at the stated tyrosine residue and panel f) t-ErbB2 normalized to actin whereas panel g) shows the ratio of p-ErbB2 (Y1221/1222) to t-ErbB2. N = 5; Mean±SD. Asterisk (*) indicates significantly different (p<0.05) mean values from normal non-diabetic rats (C) whereas hash (#) indicates significantly different mean values (p<0.05) from diabetic rats (D).
    P Her2 Erbb2 Antibody, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/p her2 erbb2 antibody/product/Cell Signaling Technology Inc
    Average 94 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    p her2 erbb2 antibody - by Bioz Stars, 2024-07
    94/100 stars

    Images

    1) Product Images from "Activation of ErbB2 and Downstream Signalling via Rho Kinases and ERK1/2 Contributes to Diabetes-Induced Vascular Dysfunction"

    Article Title: Activation of ErbB2 and Downstream Signalling via Rho Kinases and ERK1/2 Contributes to Diabetes-Induced Vascular Dysfunction

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0067813

    Panel a) is a representative Western blot showing the levels of phosphorylated ErbB2 (p-ErbB2) at the indicated tyrosines Y1221/1222, Y1248 (detected by separate antibodies labeled as Y1248 a and Y1248b, Y877, total (t-) ErbB2 and β-actin in the isolated mesenteric bed from normal controls (C), diabetic (D) and diabetic animals treated for 4 weeks with AG825 (1 mg/kg/ alt-diem ; +AG825). Panels b-e) are densitometry histograms showing levels of phosphorylated EGFR at the stated tyrosine residue and panel f) t-ErbB2 normalized to actin whereas panel g) shows the ratio of p-ErbB2 (Y1221/1222) to t-ErbB2. N = 5; Mean±SD. Asterisk (*) indicates significantly different (p<0.05) mean values from normal non-diabetic rats (C) whereas hash (#) indicates significantly different mean values (p<0.05) from diabetic rats (D).
    Figure Legend Snippet: Panel a) is a representative Western blot showing the levels of phosphorylated ErbB2 (p-ErbB2) at the indicated tyrosines Y1221/1222, Y1248 (detected by separate antibodies labeled as Y1248 a and Y1248b, Y877, total (t-) ErbB2 and β-actin in the isolated mesenteric bed from normal controls (C), diabetic (D) and diabetic animals treated for 4 weeks with AG825 (1 mg/kg/ alt-diem ; +AG825). Panels b-e) are densitometry histograms showing levels of phosphorylated EGFR at the stated tyrosine residue and panel f) t-ErbB2 normalized to actin whereas panel g) shows the ratio of p-ErbB2 (Y1221/1222) to t-ErbB2. N = 5; Mean±SD. Asterisk (*) indicates significantly different (p<0.05) mean values from normal non-diabetic rats (C) whereas hash (#) indicates significantly different mean values (p<0.05) from diabetic rats (D).

    Techniques Used: Western Blot, Labeling, Isolation

    Panel a) and c) are represenatative Western Blots following immunoprecipitations (IP) with either total-erbB2 or total-EGFR antibody and subsequent immunoblotting (IB) with both antibodies individually. Panel b) and d) represents the mean ratio of erbB2/EGFR dimers as assessed by densitometry for non-diabetic controls, (C), diabetic (D) and diabetic animals chronically treated with AG825 (+AG825) or AG1478 (+ AG1478) (both at dose of 1 mg/kg/ alt-diem ). N = 4; Asterisk (*) indicates significantly different (p<0.05) mean values from normal non-diabetic rats (C) whereas hash (#) indicates significantly different mean values (p<0.05) from diabetic rats (D).
    Figure Legend Snippet: Panel a) and c) are represenatative Western Blots following immunoprecipitations (IP) with either total-erbB2 or total-EGFR antibody and subsequent immunoblotting (IB) with both antibodies individually. Panel b) and d) represents the mean ratio of erbB2/EGFR dimers as assessed by densitometry for non-diabetic controls, (C), diabetic (D) and diabetic animals chronically treated with AG825 (+AG825) or AG1478 (+ AG1478) (both at dose of 1 mg/kg/ alt-diem ). N = 4; Asterisk (*) indicates significantly different (p<0.05) mean values from normal non-diabetic rats (C) whereas hash (#) indicates significantly different mean values (p<0.05) from diabetic rats (D).

    Techniques Used: Western Blot

    A) Panel i) is a representative Western blot showing total (t-) or phosphorylated (p) levels of the stated proteins in VSMC grown in normal (5.5mM) D-glucose (NG), high glucose (25.5mM D-glucose; HG) or HG cotreated with increasing micromolar doses of AG825 (+ AG825). Panels ii-viii) are densitometry histograms showing total (t-) or phosphorylated (p-) levels of the stated proteins normalized to actin. B) Panel i) is a representative Western blot showing total (t-) or phosphorylated (p) levels of the stated proteins in VSMC grown in high glucose (25.5mM D-glucose; HG) or HG cotreated with increasing doses of anti-ErbB2 siRNA (ErbB2 siRNA) or non-targeting control siRNA (C siRNA). Panels ii-viii) are densitometry histograms showing total (t-) or phosphorylated (p-) levels of the stated proteins normalized to actin. N = 5; Mean±SD. Asterisk (*) indicates significantly different (p<0.05) mean values from normal non-diabetic rats (C) whereas hash (#) indicates significantly different mean values (p<0.05) from diabetic rats (D).
    Figure Legend Snippet: A) Panel i) is a representative Western blot showing total (t-) or phosphorylated (p) levels of the stated proteins in VSMC grown in normal (5.5mM) D-glucose (NG), high glucose (25.5mM D-glucose; HG) or HG cotreated with increasing micromolar doses of AG825 (+ AG825). Panels ii-viii) are densitometry histograms showing total (t-) or phosphorylated (p-) levels of the stated proteins normalized to actin. B) Panel i) is a representative Western blot showing total (t-) or phosphorylated (p) levels of the stated proteins in VSMC grown in high glucose (25.5mM D-glucose; HG) or HG cotreated with increasing doses of anti-ErbB2 siRNA (ErbB2 siRNA) or non-targeting control siRNA (C siRNA). Panels ii-viii) are densitometry histograms showing total (t-) or phosphorylated (p-) levels of the stated proteins normalized to actin. N = 5; Mean±SD. Asterisk (*) indicates significantly different (p<0.05) mean values from normal non-diabetic rats (C) whereas hash (#) indicates significantly different mean values (p<0.05) from diabetic rats (D).

    Techniques Used: Western Blot

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  • 86
    Cell Signaling Technology Inc p her2 erbb2
    ( A, B ) Control and <t>ErbB2-overexpressing</t> MCF-7 cells (high levels) were treated with CDK4i or DMSO 12 h after HRG stimulation and then harvested at 24 h post-HRG stimulation. Western blotting results (A) showing p-ErbB2, p-RB (Ser 807/811 ), p-RB (Thr 373 ), RB, p-AKT (Thr 308 ), AKT, p-ERK (Thr 202 /Thr 204 ), ERK, c-Myc, cyclin D1, and p27 expression (in control cells, i.e., DMSO treatment) after normalization with GAPDH (B). ( C ) Control and ErbB2-overexpressing cells (moderate and high levels) were individually treated with 250 nM CDK4i or DMSO (no treatment) 12 h after stimulation with 10 nM HRG, and images were acquired every 20 min until 72 h after HRG stimulation. Scale bar: 100 µm. ( D ) Images in (C) were analyzed to quantitatively determine the proportion (%) of each cell-cycle phase in the entire set of images; n = 3. ( E ) Cells that completed the M/G1 transition within 72 h out of the n = 100 individually tracked cells were used. Control cells; CDK4i n = 92, DMSO n = 94, ErbB2 OE-Moderate cells; CDK4i n = 53, DMSO n = 88, ErbB2 OE-High; CDK4i n = 81, DMSO n = 91, with the time to G1/S transition plotted on the x-axis and the time to M/G1 transition plotted on the y-axis. The slope (s) and intercept (i) obtained from the regression equation are shown. The red and blue colors indicate the CDK4i and DMSO treatments, respectively. The mean ± SE time taken to reach the respective time to G1/S transition is shown at the bottom of the graph. ( F ) Using the same data as in E, the time to reach the G1/S transition (G1), the duration of the S/G2/M phase (SG2M), and the overall cell-cycle length (G1+SG2M) were assessed for differences in mean time between the CDK4i and DMSO conditions. ( G ) The number of nuclei increased from 16 h to 72 h after HRG stimulation; n = 3). In (B) and (G), * P < 0.05 (Tukey test).
    P Her2 Erbb2, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    86
    Cell Signaling Technology Inc p her2 erbb2 tyr877
    ( A, B ) Control and <t>ErbB2-overexpressing</t> MCF-7 cells (high levels) were treated with CDK4i or DMSO 12 h after HRG stimulation and then harvested at 24 h post-HRG stimulation. Western blotting results (A) showing p-ErbB2, p-RB (Ser 807/811 ), p-RB (Thr 373 ), RB, p-AKT (Thr 308 ), AKT, p-ERK (Thr 202 /Thr 204 ), ERK, c-Myc, cyclin D1, and p27 expression (in control cells, i.e., DMSO treatment) after normalization with GAPDH (B). ( C ) Control and ErbB2-overexpressing cells (moderate and high levels) were individually treated with 250 nM CDK4i or DMSO (no treatment) 12 h after stimulation with 10 nM HRG, and images were acquired every 20 min until 72 h after HRG stimulation. Scale bar: 100 µm. ( D ) Images in (C) were analyzed to quantitatively determine the proportion (%) of each cell-cycle phase in the entire set of images; n = 3. ( E ) Cells that completed the M/G1 transition within 72 h out of the n = 100 individually tracked cells were used. Control cells; CDK4i n = 92, DMSO n = 94, ErbB2 OE-Moderate cells; CDK4i n = 53, DMSO n = 88, ErbB2 OE-High; CDK4i n = 81, DMSO n = 91, with the time to G1/S transition plotted on the x-axis and the time to M/G1 transition plotted on the y-axis. The slope (s) and intercept (i) obtained from the regression equation are shown. The red and blue colors indicate the CDK4i and DMSO treatments, respectively. The mean ± SE time taken to reach the respective time to G1/S transition is shown at the bottom of the graph. ( F ) Using the same data as in E, the time to reach the G1/S transition (G1), the duration of the S/G2/M phase (SG2M), and the overall cell-cycle length (G1+SG2M) were assessed for differences in mean time between the CDK4i and DMSO conditions. ( G ) The number of nuclei increased from 16 h to 72 h after HRG stimulation; n = 3). In (B) and (G), * P < 0.05 (Tukey test).
    P Her2 Erbb2 Tyr877, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    96
    Cell Signaling Technology Inc p-her2/erbb2 y122
    ( A, B ) Control and <t>ErbB2-overexpressing</t> MCF-7 cells (high levels) were treated with CDK4i or DMSO 12 h after HRG stimulation and then harvested at 24 h post-HRG stimulation. Western blotting results (A) showing p-ErbB2, p-RB (Ser 807/811 ), p-RB (Thr 373 ), RB, p-AKT (Thr 308 ), AKT, p-ERK (Thr 202 /Thr 204 ), ERK, c-Myc, cyclin D1, and p27 expression (in control cells, i.e., DMSO treatment) after normalization with GAPDH (B). ( C ) Control and ErbB2-overexpressing cells (moderate and high levels) were individually treated with 250 nM CDK4i or DMSO (no treatment) 12 h after stimulation with 10 nM HRG, and images were acquired every 20 min until 72 h after HRG stimulation. Scale bar: 100 µm. ( D ) Images in (C) were analyzed to quantitatively determine the proportion (%) of each cell-cycle phase in the entire set of images; n = 3. ( E ) Cells that completed the M/G1 transition within 72 h out of the n = 100 individually tracked cells were used. Control cells; CDK4i n = 92, DMSO n = 94, ErbB2 OE-Moderate cells; CDK4i n = 53, DMSO n = 88, ErbB2 OE-High; CDK4i n = 81, DMSO n = 91, with the time to G1/S transition plotted on the x-axis and the time to M/G1 transition plotted on the y-axis. The slope (s) and intercept (i) obtained from the regression equation are shown. The red and blue colors indicate the CDK4i and DMSO treatments, respectively. The mean ± SE time taken to reach the respective time to G1/S transition is shown at the bottom of the graph. ( F ) Using the same data as in E, the time to reach the G1/S transition (G1), the duration of the S/G2/M phase (SG2M), and the overall cell-cycle length (G1+SG2M) were assessed for differences in mean time between the CDK4i and DMSO conditions. ( G ) The number of nuclei increased from 16 h to 72 h after HRG stimulation; n = 3). In (B) and (G), * P < 0.05 (Tukey test).
    P Her2/Erbb2 Y122, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 96/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/p-her2/erbb2 y122/product/Cell Signaling Technology Inc
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    94
    Cell Signaling Technology Inc p her2
    Clinicopathological characteristics of breast cancer patients.
    P Her2, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Cell Signaling Technology Inc p erbb2 tyr1248
    Primer sequences used for the qPCR analysis
    P Erbb2 Tyr1248, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc p her2 erbb2 antibody
    Panel a) is a representative Western blot showing the levels of phosphorylated <t>ErbB2</t> (p-ErbB2) at the indicated tyrosines Y1221/1222, Y1248 (detected by separate antibodies labeled as Y1248 a and <t>Y1248b,</t> Y877, total (t-) ErbB2 and β-actin in the isolated mesenteric bed from normal controls (C), diabetic (D) and diabetic animals treated for 4 weeks with AG825 (1 mg/kg/ alt-diem ; +AG825). Panels b-e) are densitometry histograms showing levels of phosphorylated EGFR at the stated tyrosine residue and panel f) t-ErbB2 normalized to actin whereas panel g) shows the ratio of p-ErbB2 (Y1221/1222) to t-ErbB2. N = 5; Mean±SD. Asterisk (*) indicates significantly different (p<0.05) mean values from normal non-diabetic rats (C) whereas hash (#) indicates significantly different mean values (p<0.05) from diabetic rats (D).
    P Her2 Erbb2 Antibody, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    96
    Cell Signaling Technology Inc p her2 erbb2 antibody tyr1221 1222
    Panel a) is a representative Western blot showing the levels of phosphorylated <t>ErbB2</t> (p-ErbB2) at the indicated tyrosines Y1221/1222, Y1248 (detected by separate antibodies labeled as Y1248 a and Y1248b, Y877, total (t-) ErbB2 and β-actin in the isolated mesenteric bed from normal controls (C), diabetic (D) and diabetic animals treated for 4 weeks with AG825 (1 mg/kg/ alt-diem ; +AG825). Panels b-e) are densitometry histograms showing levels of phosphorylated EGFR at the stated tyrosine residue and panel f) t-ErbB2 normalized to actin whereas panel g) shows the ratio of p-ErbB2 (Y1221/1222) to t-ErbB2. N = 5; Mean±SD. Asterisk (*) indicates significantly different (p<0.05) mean values from normal non-diabetic rats (C) whereas hash (#) indicates significantly different mean values (p<0.05) from diabetic rats (D).
    P Her2 Erbb2 Antibody Tyr1221 1222, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 96/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    ( A, B ) Control and ErbB2-overexpressing MCF-7 cells (high levels) were treated with CDK4i or DMSO 12 h after HRG stimulation and then harvested at 24 h post-HRG stimulation. Western blotting results (A) showing p-ErbB2, p-RB (Ser 807/811 ), p-RB (Thr 373 ), RB, p-AKT (Thr 308 ), AKT, p-ERK (Thr 202 /Thr 204 ), ERK, c-Myc, cyclin D1, and p27 expression (in control cells, i.e., DMSO treatment) after normalization with GAPDH (B). ( C ) Control and ErbB2-overexpressing cells (moderate and high levels) were individually treated with 250 nM CDK4i or DMSO (no treatment) 12 h after stimulation with 10 nM HRG, and images were acquired every 20 min until 72 h after HRG stimulation. Scale bar: 100 µm. ( D ) Images in (C) were analyzed to quantitatively determine the proportion (%) of each cell-cycle phase in the entire set of images; n = 3. ( E ) Cells that completed the M/G1 transition within 72 h out of the n = 100 individually tracked cells were used. Control cells; CDK4i n = 92, DMSO n = 94, ErbB2 OE-Moderate cells; CDK4i n = 53, DMSO n = 88, ErbB2 OE-High; CDK4i n = 81, DMSO n = 91, with the time to G1/S transition plotted on the x-axis and the time to M/G1 transition plotted on the y-axis. The slope (s) and intercept (i) obtained from the regression equation are shown. The red and blue colors indicate the CDK4i and DMSO treatments, respectively. The mean ± SE time taken to reach the respective time to G1/S transition is shown at the bottom of the graph. ( F ) Using the same data as in E, the time to reach the G1/S transition (G1), the duration of the S/G2/M phase (SG2M), and the overall cell-cycle length (G1+SG2M) were assessed for differences in mean time between the CDK4i and DMSO conditions. ( G ) The number of nuclei increased from 16 h to 72 h after HRG stimulation; n = 3). In (B) and (G), * P < 0.05 (Tukey test).

    Journal: bioRxiv

    Article Title: ErbB2/HER2 expression level determines CDK4-inhibitor sensitivity and cyclin D1 and c-Myc dependency at the G1/S transition

    doi: 10.1101/2024.05.09.593450

    Figure Lengend Snippet: ( A, B ) Control and ErbB2-overexpressing MCF-7 cells (high levels) were treated with CDK4i or DMSO 12 h after HRG stimulation and then harvested at 24 h post-HRG stimulation. Western blotting results (A) showing p-ErbB2, p-RB (Ser 807/811 ), p-RB (Thr 373 ), RB, p-AKT (Thr 308 ), AKT, p-ERK (Thr 202 /Thr 204 ), ERK, c-Myc, cyclin D1, and p27 expression (in control cells, i.e., DMSO treatment) after normalization with GAPDH (B). ( C ) Control and ErbB2-overexpressing cells (moderate and high levels) were individually treated with 250 nM CDK4i or DMSO (no treatment) 12 h after stimulation with 10 nM HRG, and images were acquired every 20 min until 72 h after HRG stimulation. Scale bar: 100 µm. ( D ) Images in (C) were analyzed to quantitatively determine the proportion (%) of each cell-cycle phase in the entire set of images; n = 3. ( E ) Cells that completed the M/G1 transition within 72 h out of the n = 100 individually tracked cells were used. Control cells; CDK4i n = 92, DMSO n = 94, ErbB2 OE-Moderate cells; CDK4i n = 53, DMSO n = 88, ErbB2 OE-High; CDK4i n = 81, DMSO n = 91, with the time to G1/S transition plotted on the x-axis and the time to M/G1 transition plotted on the y-axis. The slope (s) and intercept (i) obtained from the regression equation are shown. The red and blue colors indicate the CDK4i and DMSO treatments, respectively. The mean ± SE time taken to reach the respective time to G1/S transition is shown at the bottom of the graph. ( F ) Using the same data as in E, the time to reach the G1/S transition (G1), the duration of the S/G2/M phase (SG2M), and the overall cell-cycle length (G1+SG2M) were assessed for differences in mean time between the CDK4i and DMSO conditions. ( G ) The number of nuclei increased from 16 h to 72 h after HRG stimulation; n = 3). In (B) and (G), * P < 0.05 (Tukey test).

    Article Snippet: Membranes were probed overnight with antibodies against AKT (1:1000; Cell Signaling Technology; #4685), p-AKT (Thr 308 ; 1:1000; Cell Signaling Technology; #2965), ERK (1:1000; Cell Signaling Technology; #9102), p-ERK (Thr 202 Tyr 204 ; 1:1000; Cell Signaling Technology; #4370), HER2/ErbB2 (1:1000; Cell Signaling Technology; #2165), p-HER2/ErbB2 (Thr 1221/1222 ; 1:1000; Cell Signaling Technology; #2249), p-c-Fos (Ser 374 ; 1:1000; Abcam; #ab55836), c-Fos (1:200; Santa Cruz; #sc-166940), p-c-Myc (Ser 62 ; 1:1000; Abcam; #ab51156), c-Myc (1:200; Santa Cruz; #sc-40), E2F-1 (1:1000; Cell Signaling Technology; #3742), p-RB (Ser 807/811 ; 1:1000; Cell Signaling Technology; #8516), p-RB (Thr 373 ; 1:1000; Abcam; #ab52975), RB1 (1:1000; NOVUS biologicals; #SPM353), cyclin D1 (1:1000; Cell Signaling Technology; #2978), cyclin E (1:200; Santa Cruz; #sc-247), cyclin A (1:200; Santa Cruz; #sc-271682), CDK4 (1:1000; Santa Cruz; #DSC-35), CDK2 (1:1000; Santa Cruz; #D-12), P27 Kip1 (1:1000; Cell Signaling Technology; #3686), and GAPDH (1:4000; Proteintech, MA, USA; 10494-1-AP) in TBS-T including 10% blocking buffer at 4 °C.

    Techniques: Western Blot, Expressing

    ( A–G ) ( A - G ) MCF-7 cells overexpressing ErbB2 (high levels) were used. ( A, B ) The following conditions were used: treatment with 250 nM CDK4i 12 h after HRG stimulation, followed by wash/no-wash treatment 4 h later; cells were harvested 40 h after HRG stimulation. Western blotting results showing the expression of p-RB, c-Myc, cyclin D1, p27, and GAPDH. After individually normalizing the proteins relative to GAPDH, the ratio to the no-wash condition was quantified; n = 3. ( C ) Washing operations were performed under the same conditions as in (A, B); cells were fixed 40 h after HRG stimulation and immunostained with p-c-Myc and cyclin D1 antibodies together with DAPI; n = 3,859. The x-axis shows the fluorescence intensity of DAPI, and the y-axis shows the p-c-Myc/cyclin D1 ratio. A higher value on the y-axis indicates that the amount of p-c-Myc in one cell was greater than that of cyclin D1. The black circle shows the vertex of each histogram, and the red dotted line indicates the G1/S transition time point. ( D, E ) Cells were treated for 30 min with/without 64 nM c-Myc inhibitor (EN4) 11.5 h after HRG stimulation. The cells were then incubated with 250 nM CDK4i for 8 h, washed, and imaged 52 h later (72 h after HRG stimulation; D). ( E ) The percentage distributions of G1 and S/G2/M phases were examined based on the images in (D); n = 3. Scale bar: 100 µm. ( F ) Twelve hours after HRG stimulation, the cells were treated with 250 nM CDK4i or DMSO (control). mRNA levels of p27 up to 20 h after HRG stimulation were examined by qPCR. ( G ) After the cells were treated with or without EN4 under the same conditions as in (D–E), RNA was collected 8 h after treatment with inhibitors, and the mRNA levels of CDKN1B were examined by qPCR; n = 3. In (B), (E), (G), * P < 0.05 ( t -test).

    Journal: bioRxiv

    Article Title: ErbB2/HER2 expression level determines CDK4-inhibitor sensitivity and cyclin D1 and c-Myc dependency at the G1/S transition

    doi: 10.1101/2024.05.09.593450

    Figure Lengend Snippet: ( A–G ) ( A - G ) MCF-7 cells overexpressing ErbB2 (high levels) were used. ( A, B ) The following conditions were used: treatment with 250 nM CDK4i 12 h after HRG stimulation, followed by wash/no-wash treatment 4 h later; cells were harvested 40 h after HRG stimulation. Western blotting results showing the expression of p-RB, c-Myc, cyclin D1, p27, and GAPDH. After individually normalizing the proteins relative to GAPDH, the ratio to the no-wash condition was quantified; n = 3. ( C ) Washing operations were performed under the same conditions as in (A, B); cells were fixed 40 h after HRG stimulation and immunostained with p-c-Myc and cyclin D1 antibodies together with DAPI; n = 3,859. The x-axis shows the fluorescence intensity of DAPI, and the y-axis shows the p-c-Myc/cyclin D1 ratio. A higher value on the y-axis indicates that the amount of p-c-Myc in one cell was greater than that of cyclin D1. The black circle shows the vertex of each histogram, and the red dotted line indicates the G1/S transition time point. ( D, E ) Cells were treated for 30 min with/without 64 nM c-Myc inhibitor (EN4) 11.5 h after HRG stimulation. The cells were then incubated with 250 nM CDK4i for 8 h, washed, and imaged 52 h later (72 h after HRG stimulation; D). ( E ) The percentage distributions of G1 and S/G2/M phases were examined based on the images in (D); n = 3. Scale bar: 100 µm. ( F ) Twelve hours after HRG stimulation, the cells were treated with 250 nM CDK4i or DMSO (control). mRNA levels of p27 up to 20 h after HRG stimulation were examined by qPCR. ( G ) After the cells were treated with or without EN4 under the same conditions as in (D–E), RNA was collected 8 h after treatment with inhibitors, and the mRNA levels of CDKN1B were examined by qPCR; n = 3. In (B), (E), (G), * P < 0.05 ( t -test).

    Article Snippet: Membranes were probed overnight with antibodies against AKT (1:1000; Cell Signaling Technology; #4685), p-AKT (Thr 308 ; 1:1000; Cell Signaling Technology; #2965), ERK (1:1000; Cell Signaling Technology; #9102), p-ERK (Thr 202 Tyr 204 ; 1:1000; Cell Signaling Technology; #4370), HER2/ErbB2 (1:1000; Cell Signaling Technology; #2165), p-HER2/ErbB2 (Thr 1221/1222 ; 1:1000; Cell Signaling Technology; #2249), p-c-Fos (Ser 374 ; 1:1000; Abcam; #ab55836), c-Fos (1:200; Santa Cruz; #sc-166940), p-c-Myc (Ser 62 ; 1:1000; Abcam; #ab51156), c-Myc (1:200; Santa Cruz; #sc-40), E2F-1 (1:1000; Cell Signaling Technology; #3742), p-RB (Ser 807/811 ; 1:1000; Cell Signaling Technology; #8516), p-RB (Thr 373 ; 1:1000; Abcam; #ab52975), RB1 (1:1000; NOVUS biologicals; #SPM353), cyclin D1 (1:1000; Cell Signaling Technology; #2978), cyclin E (1:200; Santa Cruz; #sc-247), cyclin A (1:200; Santa Cruz; #sc-271682), CDK4 (1:1000; Santa Cruz; #DSC-35), CDK2 (1:1000; Santa Cruz; #D-12), P27 Kip1 (1:1000; Cell Signaling Technology; #3686), and GAPDH (1:4000; Proteintech, MA, USA; 10494-1-AP) in TBS-T including 10% blocking buffer at 4 °C.

    Techniques: Western Blot, Expressing, Fluorescence, Incubation

    (A) In WT MCF-7 cells, the G1/S transition is facilitated by the regulation of cyclin D1/CDK4 via ERK and AKT activities. Conversely, CDK4 inhibition leads to reduced signaling activity of AKT and ERK due to ErbB2 degradation via Hsp90 dis-colocalization, leading to attenuated transcriptional activity of c-Myc activated by epigenetic changes. Consequently, a subpopulation of cells that is unresponsive to CDK4is exhibits a prolonged G1/S transition time. ( B ) In MCF-7 cells expressing high levels of high ErbB2, increased AKT activity leads to increased c-Myc expression. Subsequently, the expression of p27, which is critical for the stability of cyclin D1/CDK4 during the G1 phase, is suppressed by c-Myc, resulting in a slightly delayed G1/S entry compared to that in wild-type cells. Conversely, following CDK4 inhibition, most cells exhibit a cell-cycle arrest. This arrest is reversibly maintained by the strong transcriptional activity of c-Myc, ultimately sustaining survival signals.

    Journal: bioRxiv

    Article Title: ErbB2/HER2 expression level determines CDK4-inhibitor sensitivity and cyclin D1 and c-Myc dependency at the G1/S transition

    doi: 10.1101/2024.05.09.593450

    Figure Lengend Snippet: (A) In WT MCF-7 cells, the G1/S transition is facilitated by the regulation of cyclin D1/CDK4 via ERK and AKT activities. Conversely, CDK4 inhibition leads to reduced signaling activity of AKT and ERK due to ErbB2 degradation via Hsp90 dis-colocalization, leading to attenuated transcriptional activity of c-Myc activated by epigenetic changes. Consequently, a subpopulation of cells that is unresponsive to CDK4is exhibits a prolonged G1/S transition time. ( B ) In MCF-7 cells expressing high levels of high ErbB2, increased AKT activity leads to increased c-Myc expression. Subsequently, the expression of p27, which is critical for the stability of cyclin D1/CDK4 during the G1 phase, is suppressed by c-Myc, resulting in a slightly delayed G1/S entry compared to that in wild-type cells. Conversely, following CDK4 inhibition, most cells exhibit a cell-cycle arrest. This arrest is reversibly maintained by the strong transcriptional activity of c-Myc, ultimately sustaining survival signals.

    Article Snippet: Membranes were probed overnight with antibodies against AKT (1:1000; Cell Signaling Technology; #4685), p-AKT (Thr 308 ; 1:1000; Cell Signaling Technology; #2965), ERK (1:1000; Cell Signaling Technology; #9102), p-ERK (Thr 202 Tyr 204 ; 1:1000; Cell Signaling Technology; #4370), HER2/ErbB2 (1:1000; Cell Signaling Technology; #2165), p-HER2/ErbB2 (Thr 1221/1222 ; 1:1000; Cell Signaling Technology; #2249), p-c-Fos (Ser 374 ; 1:1000; Abcam; #ab55836), c-Fos (1:200; Santa Cruz; #sc-166940), p-c-Myc (Ser 62 ; 1:1000; Abcam; #ab51156), c-Myc (1:200; Santa Cruz; #sc-40), E2F-1 (1:1000; Cell Signaling Technology; #3742), p-RB (Ser 807/811 ; 1:1000; Cell Signaling Technology; #8516), p-RB (Thr 373 ; 1:1000; Abcam; #ab52975), RB1 (1:1000; NOVUS biologicals; #SPM353), cyclin D1 (1:1000; Cell Signaling Technology; #2978), cyclin E (1:200; Santa Cruz; #sc-247), cyclin A (1:200; Santa Cruz; #sc-271682), CDK4 (1:1000; Santa Cruz; #DSC-35), CDK2 (1:1000; Santa Cruz; #D-12), P27 Kip1 (1:1000; Cell Signaling Technology; #3686), and GAPDH (1:4000; Proteintech, MA, USA; 10494-1-AP) in TBS-T including 10% blocking buffer at 4 °C.

    Techniques: Inhibition, Activity Assay, Expressing

    Clinicopathological characteristics of breast cancer patients.

    Journal: International Journal of Molecular Sciences

    Article Title: Tumorigenicity of EGFR- and/or HER2-Positive Breast Cancers Is Mediated by Recruitment of Tumor-Associated Macrophages

    doi: 10.3390/ijms24021443

    Figure Lengend Snippet: Clinicopathological characteristics of breast cancer patients.

    Article Snippet: Membranes were blocked with skim milk at RT for 1 h and incubated with the following primary antibodies at 4 °C overnight (O/N): β-actin (Abfrontier, Seoul, Republic of Korea, LF-PA0207, 1:2000), t-EGFR (Abcam, Waltham, MA, USA, ab52894, 1:10,000), p -EGFR (Abcam, ab40815, 1:5000), t-HER2 (Santa Cruz Biotechnology, CA, USA, sc33684, 1:5000), p -HER2 (CST, Danvers, MA, USA, 2241S, 1:1000), and CD11b (Abcam, ab52478, 1:1000).

    Techniques:

    Co-expression of EGFR and HER2 correlates with poor survival in breast cancer patients. ( A ) Disease-free survival rates were compared between EGFR+/HER2-/ER- ( n = 92) and EGFR+/HER2+/ER- ( n = 23) patients ( p = 0.0229). ( B ) Overall survival rates were compared between EGFR+/HER2-/ER- ( n = 92) and EGFR+/HER2+/ER- ( n = 23) patients ( p = 0.019).

    Journal: International Journal of Molecular Sciences

    Article Title: Tumorigenicity of EGFR- and/or HER2-Positive Breast Cancers Is Mediated by Recruitment of Tumor-Associated Macrophages

    doi: 10.3390/ijms24021443

    Figure Lengend Snippet: Co-expression of EGFR and HER2 correlates with poor survival in breast cancer patients. ( A ) Disease-free survival rates were compared between EGFR+/HER2-/ER- ( n = 92) and EGFR+/HER2+/ER- ( n = 23) patients ( p = 0.0229). ( B ) Overall survival rates were compared between EGFR+/HER2-/ER- ( n = 92) and EGFR+/HER2+/ER- ( n = 23) patients ( p = 0.019).

    Article Snippet: Membranes were blocked with skim milk at RT for 1 h and incubated with the following primary antibodies at 4 °C overnight (O/N): β-actin (Abfrontier, Seoul, Republic of Korea, LF-PA0207, 1:2000), t-EGFR (Abcam, Waltham, MA, USA, ab52894, 1:10,000), p -EGFR (Abcam, ab40815, 1:5000), t-HER2 (Santa Cruz Biotechnology, CA, USA, sc33684, 1:5000), p -HER2 (CST, Danvers, MA, USA, 2241S, 1:1000), and CD11b (Abcam, ab52478, 1:1000).

    Techniques: Expressing

    Co-expression of EGFR and HER2 promotes invasion and proliferation. ( A ) Transcript levels of EGFR and HER2 in Vec and HER2-overexpressed MDA-MB231 cells were analyzed using quantitative reverse transcription polymerase chain reaction (RT-qPCR). Values were normalized to ACTB . ( B ) Levels of protein expression were analyzed using Western blotting with indicated antibodies. β-actin was used as a loading control. ( C ) Invasion assays were performed using MDA-MB231 Vec and HER2-overexpressed cells as described in the method section. The scale bar represents 200 μm. ( D ) Cell cycle analysis was performed using MDA-MB231 Vec and HER2-overexpressed cells. * p < 0.05.

    Journal: International Journal of Molecular Sciences

    Article Title: Tumorigenicity of EGFR- and/or HER2-Positive Breast Cancers Is Mediated by Recruitment of Tumor-Associated Macrophages

    doi: 10.3390/ijms24021443

    Figure Lengend Snippet: Co-expression of EGFR and HER2 promotes invasion and proliferation. ( A ) Transcript levels of EGFR and HER2 in Vec and HER2-overexpressed MDA-MB231 cells were analyzed using quantitative reverse transcription polymerase chain reaction (RT-qPCR). Values were normalized to ACTB . ( B ) Levels of protein expression were analyzed using Western blotting with indicated antibodies. β-actin was used as a loading control. ( C ) Invasion assays were performed using MDA-MB231 Vec and HER2-overexpressed cells as described in the method section. The scale bar represents 200 μm. ( D ) Cell cycle analysis was performed using MDA-MB231 Vec and HER2-overexpressed cells. * p < 0.05.

    Article Snippet: Membranes were blocked with skim milk at RT for 1 h and incubated with the following primary antibodies at 4 °C overnight (O/N): β-actin (Abfrontier, Seoul, Republic of Korea, LF-PA0207, 1:2000), t-EGFR (Abcam, Waltham, MA, USA, ab52894, 1:10,000), p -EGFR (Abcam, ab40815, 1:5000), t-HER2 (Santa Cruz Biotechnology, CA, USA, sc33684, 1:5000), p -HER2 (CST, Danvers, MA, USA, 2241S, 1:1000), and CD11b (Abcam, ab52478, 1:1000).

    Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Quantitative RT-PCR, Western Blot, Cell Cycle Assay

    Cytokine secretion profile is altered by HER2 overexpression. ( A ) Human cytokine array was performed using conditioned culture media of MDA-MB231 Vec and HER2-overexpressed cells. ( B – E ) Transcript levels of CCL2 , CCL5 , IL-6 , and IL-8 in Vec and HER2-overexpressed MDA-MB231 cells were analyzed using RT-qPCR. Values were normalized to ACTB . * p < 0.05.

    Journal: International Journal of Molecular Sciences

    Article Title: Tumorigenicity of EGFR- and/or HER2-Positive Breast Cancers Is Mediated by Recruitment of Tumor-Associated Macrophages

    doi: 10.3390/ijms24021443

    Figure Lengend Snippet: Cytokine secretion profile is altered by HER2 overexpression. ( A ) Human cytokine array was performed using conditioned culture media of MDA-MB231 Vec and HER2-overexpressed cells. ( B – E ) Transcript levels of CCL2 , CCL5 , IL-6 , and IL-8 in Vec and HER2-overexpressed MDA-MB231 cells were analyzed using RT-qPCR. Values were normalized to ACTB . * p < 0.05.

    Article Snippet: Membranes were blocked with skim milk at RT for 1 h and incubated with the following primary antibodies at 4 °C overnight (O/N): β-actin (Abfrontier, Seoul, Republic of Korea, LF-PA0207, 1:2000), t-EGFR (Abcam, Waltham, MA, USA, ab52894, 1:10,000), p -EGFR (Abcam, ab40815, 1:5000), t-HER2 (Santa Cruz Biotechnology, CA, USA, sc33684, 1:5000), p -HER2 (CST, Danvers, MA, USA, 2241S, 1:1000), and CD11b (Abcam, ab52478, 1:1000).

    Techniques: Over Expression, Quantitative RT-PCR

    CCL2 expression is downregulated by neratinib. ( A , B ) Each cell line was treated with 5 μM neratinib for 24 h. ( A ) Transcript levels of CCL2 were analyzed using RT-qPCR. Values were normalized to ACTB . ( B ) ELISA was performed using conditioned culture media of Vec and HER2-overexpressed MDA-MB231 cells. ( C ) Levels of protein expression were analyzed using Western blotting with indicated antibodies. β-actin was used as a loading control. ( D ) ELISA was performed using conditioned culture media of MDA453 and BT474 cells transfected with scrambled or HER2-specific siRNA. * p < 0.05; ** p < 0.01; ϕ p < 0.05.

    Journal: International Journal of Molecular Sciences

    Article Title: Tumorigenicity of EGFR- and/or HER2-Positive Breast Cancers Is Mediated by Recruitment of Tumor-Associated Macrophages

    doi: 10.3390/ijms24021443

    Figure Lengend Snippet: CCL2 expression is downregulated by neratinib. ( A , B ) Each cell line was treated with 5 μM neratinib for 24 h. ( A ) Transcript levels of CCL2 were analyzed using RT-qPCR. Values were normalized to ACTB . ( B ) ELISA was performed using conditioned culture media of Vec and HER2-overexpressed MDA-MB231 cells. ( C ) Levels of protein expression were analyzed using Western blotting with indicated antibodies. β-actin was used as a loading control. ( D ) ELISA was performed using conditioned culture media of MDA453 and BT474 cells transfected with scrambled or HER2-specific siRNA. * p < 0.05; ** p < 0.01; ϕ p < 0.05.

    Article Snippet: Membranes were blocked with skim milk at RT for 1 h and incubated with the following primary antibodies at 4 °C overnight (O/N): β-actin (Abfrontier, Seoul, Republic of Korea, LF-PA0207, 1:2000), t-EGFR (Abcam, Waltham, MA, USA, ab52894, 1:10,000), p -EGFR (Abcam, ab40815, 1:5000), t-HER2 (Santa Cruz Biotechnology, CA, USA, sc33684, 1:5000), p -HER2 (CST, Danvers, MA, USA, 2241S, 1:1000), and CD11b (Abcam, ab52478, 1:1000).

    Techniques: Expressing, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Western Blot, Transfection

    CCL2 mediates motility of TAMs. ( A ) Immunofluorescence confocal microscopy was performed using M0 and M2-differentiated THP-1 cells. Anti-CD11b primary antibody and Alexa Fluor 488-conjugated secondary antibody were used for staining. The nucleus was stained with DAPI. ( B ) Upper, levels of CD-11b and CD163 protein expression were analyzed using Western blotting with whole cell lysates from M0 and M2-differentiated THP-1 cells with indicated antibodies. β-actin was used as a loading control. Lower, transcript levels of CD11b in M0 and M2-differentiated THP-1 cells were analyzed using RT-qPCR. Values were normalized to ACTB. ( C ) Migration assays were performed as described in the method section. ( D ) Cytokine arrays were performed using whole cell lysates of control and CCL2-treated M2-differentiated THP-1 cells. ( E ) Migration assays were performed with M2-differentiated THP-1 cells treated with conditioned culture media from MDA-MB231 Vec and HER2-overexpressed cells. The scale bar represents 200 μm. ( F ) RT-qPCR showing transcript levels of IL-8 and IL-1B in M2-differentiated THP-1 cells treated with conditioned culture media from MDA-MB231 Vec and HER2-overexpressed cells. Values were normalized to ACTB . * p < 0.05.

    Journal: International Journal of Molecular Sciences

    Article Title: Tumorigenicity of EGFR- and/or HER2-Positive Breast Cancers Is Mediated by Recruitment of Tumor-Associated Macrophages

    doi: 10.3390/ijms24021443

    Figure Lengend Snippet: CCL2 mediates motility of TAMs. ( A ) Immunofluorescence confocal microscopy was performed using M0 and M2-differentiated THP-1 cells. Anti-CD11b primary antibody and Alexa Fluor 488-conjugated secondary antibody were used for staining. The nucleus was stained with DAPI. ( B ) Upper, levels of CD-11b and CD163 protein expression were analyzed using Western blotting with whole cell lysates from M0 and M2-differentiated THP-1 cells with indicated antibodies. β-actin was used as a loading control. Lower, transcript levels of CD11b in M0 and M2-differentiated THP-1 cells were analyzed using RT-qPCR. Values were normalized to ACTB. ( C ) Migration assays were performed as described in the method section. ( D ) Cytokine arrays were performed using whole cell lysates of control and CCL2-treated M2-differentiated THP-1 cells. ( E ) Migration assays were performed with M2-differentiated THP-1 cells treated with conditioned culture media from MDA-MB231 Vec and HER2-overexpressed cells. The scale bar represents 200 μm. ( F ) RT-qPCR showing transcript levels of IL-8 and IL-1B in M2-differentiated THP-1 cells treated with conditioned culture media from MDA-MB231 Vec and HER2-overexpressed cells. Values were normalized to ACTB . * p < 0.05.

    Article Snippet: Membranes were blocked with skim milk at RT for 1 h and incubated with the following primary antibodies at 4 °C overnight (O/N): β-actin (Abfrontier, Seoul, Republic of Korea, LF-PA0207, 1:2000), t-EGFR (Abcam, Waltham, MA, USA, ab52894, 1:10,000), p -EGFR (Abcam, ab40815, 1:5000), t-HER2 (Santa Cruz Biotechnology, CA, USA, sc33684, 1:5000), p -HER2 (CST, Danvers, MA, USA, 2241S, 1:1000), and CD11b (Abcam, ab52478, 1:1000).

    Techniques: Immunofluorescence, Confocal Microscopy, Staining, Expressing, Western Blot, Quantitative RT-PCR, Migration

    TAMs recruited into HER2-overexpressed tumors. ( A ) A schematic diagram illustrating the xenograft study in this figure: MDA-MB231 Vec or HER2-overexpressed cells were injected into the 2nd fat pad of NOD/SCID mice. Recruited TAMs were monitored after 12 weeks. ( B ) Recruited TAMs were analyzed using CD163 staining.

    Journal: International Journal of Molecular Sciences

    Article Title: Tumorigenicity of EGFR- and/or HER2-Positive Breast Cancers Is Mediated by Recruitment of Tumor-Associated Macrophages

    doi: 10.3390/ijms24021443

    Figure Lengend Snippet: TAMs recruited into HER2-overexpressed tumors. ( A ) A schematic diagram illustrating the xenograft study in this figure: MDA-MB231 Vec or HER2-overexpressed cells were injected into the 2nd fat pad of NOD/SCID mice. Recruited TAMs were monitored after 12 weeks. ( B ) Recruited TAMs were analyzed using CD163 staining.

    Article Snippet: Membranes were blocked with skim milk at RT for 1 h and incubated with the following primary antibodies at 4 °C overnight (O/N): β-actin (Abfrontier, Seoul, Republic of Korea, LF-PA0207, 1:2000), t-EGFR (Abcam, Waltham, MA, USA, ab52894, 1:10,000), p -EGFR (Abcam, ab40815, 1:5000), t-HER2 (Santa Cruz Biotechnology, CA, USA, sc33684, 1:5000), p -HER2 (CST, Danvers, MA, USA, 2241S, 1:1000), and CD11b (Abcam, ab52478, 1:1000).

    Techniques: Injection, Staining

    Primer sequences used for the qPCR analysis

    Journal: Drug Design, Development and Therapy

    Article Title: Dysregulation of Neuregulin-1/ErbB signaling in the hippocampus of rats after administration of doxorubicin

    doi: 10.2147/DDDT.S151511

    Figure Lengend Snippet: Primer sequences used for the qPCR analysis

    Article Snippet: The following antibodies and concentrations were used over night at 4°C; NRG1 (Santa Cruz Biotechnology Inc., Dallas, TX, USA, sc-28916; 1:400), ErbB4 (Santa Cruz Biotechnology Inc., sc-283; 1:500), ErbB2 (Cell Signaling, Danvers, MA, USA, 2165; 1:1,500), p-ErbB4 (Tyr1056) (Santa Cruz Biotechnology Inc., sc33040; 1:300), p-ErbB2 (Tyr1248) (Cell Signaling, 2247; 1:1,500), p-Akt (Ser473) (Cell Signaling, 4060; 1:3,000), p-ERK (Thr202/Tyr204) (Cell Signaling, 4695; 1:2,000), caspase-3 (Abcam, Cambridge, UK, ab4051; 1:500), and β-actin (Proteintech, Rosemont, IL, USA, 66009-1-Ig; 1:4,000).

    Techniques: Amplification

    Effect of Dox on gene expression of ErbB2, ErbB4, and the ratio of pErbB4/ErbB4 and pErbB2/ErbB2 in the hippocampus. Notes: ErbB4 mRNA expression ( A ); pErbB4/ErbB4 ratio ( B ); ErbB2 mRNA expression ( C ); and pErbB2/ErbB2 ratio ( D ). Data are expressed as mean ± SEM (n=6–7). * p <0.05 and ** p <0.01 compared to the control group. Abbreviations: Dox, doxorubicin; DoxS, doxorubicin administration for short time; DoxL, doxorubicin administration for long time; SEM, standard error of the mean.

    Journal: Drug Design, Development and Therapy

    Article Title: Dysregulation of Neuregulin-1/ErbB signaling in the hippocampus of rats after administration of doxorubicin

    doi: 10.2147/DDDT.S151511

    Figure Lengend Snippet: Effect of Dox on gene expression of ErbB2, ErbB4, and the ratio of pErbB4/ErbB4 and pErbB2/ErbB2 in the hippocampus. Notes: ErbB4 mRNA expression ( A ); pErbB4/ErbB4 ratio ( B ); ErbB2 mRNA expression ( C ); and pErbB2/ErbB2 ratio ( D ). Data are expressed as mean ± SEM (n=6–7). * p <0.05 and ** p <0.01 compared to the control group. Abbreviations: Dox, doxorubicin; DoxS, doxorubicin administration for short time; DoxL, doxorubicin administration for long time; SEM, standard error of the mean.

    Article Snippet: The following antibodies and concentrations were used over night at 4°C; NRG1 (Santa Cruz Biotechnology Inc., Dallas, TX, USA, sc-28916; 1:400), ErbB4 (Santa Cruz Biotechnology Inc., sc-283; 1:500), ErbB2 (Cell Signaling, Danvers, MA, USA, 2165; 1:1,500), p-ErbB4 (Tyr1056) (Santa Cruz Biotechnology Inc., sc33040; 1:300), p-ErbB2 (Tyr1248) (Cell Signaling, 2247; 1:1,500), p-Akt (Ser473) (Cell Signaling, 4060; 1:3,000), p-ERK (Thr202/Tyr204) (Cell Signaling, 4695; 1:2,000), caspase-3 (Abcam, Cambridge, UK, ab4051; 1:500), and β-actin (Proteintech, Rosemont, IL, USA, 66009-1-Ig; 1:4,000).

    Techniques: Expressing

    Panel a) is a representative Western blot showing the levels of phosphorylated ErbB2 (p-ErbB2) at the indicated tyrosines Y1221/1222, Y1248 (detected by separate antibodies labeled as Y1248 a and Y1248b, Y877, total (t-) ErbB2 and β-actin in the isolated mesenteric bed from normal controls (C), diabetic (D) and diabetic animals treated for 4 weeks with AG825 (1 mg/kg/ alt-diem ; +AG825). Panels b-e) are densitometry histograms showing levels of phosphorylated EGFR at the stated tyrosine residue and panel f) t-ErbB2 normalized to actin whereas panel g) shows the ratio of p-ErbB2 (Y1221/1222) to t-ErbB2. N = 5; Mean±SD. Asterisk (*) indicates significantly different (p<0.05) mean values from normal non-diabetic rats (C) whereas hash (#) indicates significantly different mean values (p<0.05) from diabetic rats (D).

    Journal: PLoS ONE

    Article Title: Activation of ErbB2 and Downstream Signalling via Rho Kinases and ERK1/2 Contributes to Diabetes-Induced Vascular Dysfunction

    doi: 10.1371/journal.pone.0067813

    Figure Lengend Snippet: Panel a) is a representative Western blot showing the levels of phosphorylated ErbB2 (p-ErbB2) at the indicated tyrosines Y1221/1222, Y1248 (detected by separate antibodies labeled as Y1248 a and Y1248b, Y877, total (t-) ErbB2 and β-actin in the isolated mesenteric bed from normal controls (C), diabetic (D) and diabetic animals treated for 4 weeks with AG825 (1 mg/kg/ alt-diem ; +AG825). Panels b-e) are densitometry histograms showing levels of phosphorylated EGFR at the stated tyrosine residue and panel f) t-ErbB2 normalized to actin whereas panel g) shows the ratio of p-ErbB2 (Y1221/1222) to t-ErbB2. N = 5; Mean±SD. Asterisk (*) indicates significantly different (p<0.05) mean values from normal non-diabetic rats (C) whereas hash (#) indicates significantly different mean values (p<0.05) from diabetic rats (D).

    Article Snippet: The following antibodies from Cell Signaling (USA) were used in this study: t-Her2/ErbB2-Antibody (29D8) (rabbit) Cat. No. 2165, p-Her2/ErbB2-Antibody (Tyr877) (rabbit) Cat. No. 2241, p-Her2/ErbB2-Antibody (Tyr1248; labeled in figures as Y1248a) (rabbit) Cat. No. 2247, p-Her2/ErbB2-Antibody (Tyr1248/EGFR Tyr1173; labelled as Y1248b) (rabbit) Cat. No. 2244, p-Her2/ErbB2-Antibody (Tyr1221/1222) (rabbit) Cat. No. 2243, p-ERK1/2 (p44/42 MAP Kinase, Thr202/Tyr204) Antibody (rabbit) Cat. No. 9101, : t-EGFR-Antibody (rabbit) Cat. No. 2232, p-EGFR-Antibody (Tyr1068) (rabbit) Cat. No. 2234, and p-EGFR-Antibody (Tyr1086) (rabbit).

    Techniques: Western Blot, Labeling, Isolation

    Panel a) and c) are represenatative Western Blots following immunoprecipitations (IP) with either total-erbB2 or total-EGFR antibody and subsequent immunoblotting (IB) with both antibodies individually. Panel b) and d) represents the mean ratio of erbB2/EGFR dimers as assessed by densitometry for non-diabetic controls, (C), diabetic (D) and diabetic animals chronically treated with AG825 (+AG825) or AG1478 (+ AG1478) (both at dose of 1 mg/kg/ alt-diem ). N = 4; Asterisk (*) indicates significantly different (p<0.05) mean values from normal non-diabetic rats (C) whereas hash (#) indicates significantly different mean values (p<0.05) from diabetic rats (D).

    Journal: PLoS ONE

    Article Title: Activation of ErbB2 and Downstream Signalling via Rho Kinases and ERK1/2 Contributes to Diabetes-Induced Vascular Dysfunction

    doi: 10.1371/journal.pone.0067813

    Figure Lengend Snippet: Panel a) and c) are represenatative Western Blots following immunoprecipitations (IP) with either total-erbB2 or total-EGFR antibody and subsequent immunoblotting (IB) with both antibodies individually. Panel b) and d) represents the mean ratio of erbB2/EGFR dimers as assessed by densitometry for non-diabetic controls, (C), diabetic (D) and diabetic animals chronically treated with AG825 (+AG825) or AG1478 (+ AG1478) (both at dose of 1 mg/kg/ alt-diem ). N = 4; Asterisk (*) indicates significantly different (p<0.05) mean values from normal non-diabetic rats (C) whereas hash (#) indicates significantly different mean values (p<0.05) from diabetic rats (D).

    Article Snippet: The following antibodies from Cell Signaling (USA) were used in this study: t-Her2/ErbB2-Antibody (29D8) (rabbit) Cat. No. 2165, p-Her2/ErbB2-Antibody (Tyr877) (rabbit) Cat. No. 2241, p-Her2/ErbB2-Antibody (Tyr1248; labeled in figures as Y1248a) (rabbit) Cat. No. 2247, p-Her2/ErbB2-Antibody (Tyr1248/EGFR Tyr1173; labelled as Y1248b) (rabbit) Cat. No. 2244, p-Her2/ErbB2-Antibody (Tyr1221/1222) (rabbit) Cat. No. 2243, p-ERK1/2 (p44/42 MAP Kinase, Thr202/Tyr204) Antibody (rabbit) Cat. No. 9101, : t-EGFR-Antibody (rabbit) Cat. No. 2232, p-EGFR-Antibody (Tyr1068) (rabbit) Cat. No. 2234, and p-EGFR-Antibody (Tyr1086) (rabbit).

    Techniques: Western Blot

    A) Panel i) is a representative Western blot showing total (t-) or phosphorylated (p) levels of the stated proteins in VSMC grown in normal (5.5mM) D-glucose (NG), high glucose (25.5mM D-glucose; HG) or HG cotreated with increasing micromolar doses of AG825 (+ AG825). Panels ii-viii) are densitometry histograms showing total (t-) or phosphorylated (p-) levels of the stated proteins normalized to actin. B) Panel i) is a representative Western blot showing total (t-) or phosphorylated (p) levels of the stated proteins in VSMC grown in high glucose (25.5mM D-glucose; HG) or HG cotreated with increasing doses of anti-ErbB2 siRNA (ErbB2 siRNA) or non-targeting control siRNA (C siRNA). Panels ii-viii) are densitometry histograms showing total (t-) or phosphorylated (p-) levels of the stated proteins normalized to actin. N = 5; Mean±SD. Asterisk (*) indicates significantly different (p<0.05) mean values from normal non-diabetic rats (C) whereas hash (#) indicates significantly different mean values (p<0.05) from diabetic rats (D).

    Journal: PLoS ONE

    Article Title: Activation of ErbB2 and Downstream Signalling via Rho Kinases and ERK1/2 Contributes to Diabetes-Induced Vascular Dysfunction

    doi: 10.1371/journal.pone.0067813

    Figure Lengend Snippet: A) Panel i) is a representative Western blot showing total (t-) or phosphorylated (p) levels of the stated proteins in VSMC grown in normal (5.5mM) D-glucose (NG), high glucose (25.5mM D-glucose; HG) or HG cotreated with increasing micromolar doses of AG825 (+ AG825). Panels ii-viii) are densitometry histograms showing total (t-) or phosphorylated (p-) levels of the stated proteins normalized to actin. B) Panel i) is a representative Western blot showing total (t-) or phosphorylated (p) levels of the stated proteins in VSMC grown in high glucose (25.5mM D-glucose; HG) or HG cotreated with increasing doses of anti-ErbB2 siRNA (ErbB2 siRNA) or non-targeting control siRNA (C siRNA). Panels ii-viii) are densitometry histograms showing total (t-) or phosphorylated (p-) levels of the stated proteins normalized to actin. N = 5; Mean±SD. Asterisk (*) indicates significantly different (p<0.05) mean values from normal non-diabetic rats (C) whereas hash (#) indicates significantly different mean values (p<0.05) from diabetic rats (D).

    Article Snippet: The following antibodies from Cell Signaling (USA) were used in this study: t-Her2/ErbB2-Antibody (29D8) (rabbit) Cat. No. 2165, p-Her2/ErbB2-Antibody (Tyr877) (rabbit) Cat. No. 2241, p-Her2/ErbB2-Antibody (Tyr1248; labeled in figures as Y1248a) (rabbit) Cat. No. 2247, p-Her2/ErbB2-Antibody (Tyr1248/EGFR Tyr1173; labelled as Y1248b) (rabbit) Cat. No. 2244, p-Her2/ErbB2-Antibody (Tyr1221/1222) (rabbit) Cat. No. 2243, p-ERK1/2 (p44/42 MAP Kinase, Thr202/Tyr204) Antibody (rabbit) Cat. No. 9101, : t-EGFR-Antibody (rabbit) Cat. No. 2232, p-EGFR-Antibody (Tyr1068) (rabbit) Cat. No. 2234, and p-EGFR-Antibody (Tyr1086) (rabbit).

    Techniques: Western Blot

    Panel a) is a representative Western blot showing the levels of phosphorylated ErbB2 (p-ErbB2) at the indicated tyrosines Y1221/1222, Y1248 (detected by separate antibodies labeled as Y1248 a and Y1248b, Y877, total (t-) ErbB2 and β-actin in the isolated mesenteric bed from normal controls (C), diabetic (D) and diabetic animals treated for 4 weeks with AG825 (1 mg/kg/ alt-diem ; +AG825). Panels b-e) are densitometry histograms showing levels of phosphorylated EGFR at the stated tyrosine residue and panel f) t-ErbB2 normalized to actin whereas panel g) shows the ratio of p-ErbB2 (Y1221/1222) to t-ErbB2. N = 5; Mean±SD. Asterisk (*) indicates significantly different (p<0.05) mean values from normal non-diabetic rats (C) whereas hash (#) indicates significantly different mean values (p<0.05) from diabetic rats (D).

    Journal: PLoS ONE

    Article Title: Activation of ErbB2 and Downstream Signalling via Rho Kinases and ERK1/2 Contributes to Diabetes-Induced Vascular Dysfunction

    doi: 10.1371/journal.pone.0067813

    Figure Lengend Snippet: Panel a) is a representative Western blot showing the levels of phosphorylated ErbB2 (p-ErbB2) at the indicated tyrosines Y1221/1222, Y1248 (detected by separate antibodies labeled as Y1248 a and Y1248b, Y877, total (t-) ErbB2 and β-actin in the isolated mesenteric bed from normal controls (C), diabetic (D) and diabetic animals treated for 4 weeks with AG825 (1 mg/kg/ alt-diem ; +AG825). Panels b-e) are densitometry histograms showing levels of phosphorylated EGFR at the stated tyrosine residue and panel f) t-ErbB2 normalized to actin whereas panel g) shows the ratio of p-ErbB2 (Y1221/1222) to t-ErbB2. N = 5; Mean±SD. Asterisk (*) indicates significantly different (p<0.05) mean values from normal non-diabetic rats (C) whereas hash (#) indicates significantly different mean values (p<0.05) from diabetic rats (D).

    Article Snippet: The following antibodies from Cell Signaling (USA) were used in this study: t-Her2/ErbB2-Antibody (29D8) (rabbit) Cat. No. 2165, p-Her2/ErbB2-Antibody (Tyr877) (rabbit) Cat. No. 2241, p-Her2/ErbB2-Antibody (Tyr1248; labeled in figures as Y1248a) (rabbit) Cat. No. 2247, p-Her2/ErbB2-Antibody (Tyr1248/EGFR Tyr1173; labelled as Y1248b) (rabbit) Cat. No. 2244, p-Her2/ErbB2-Antibody (Tyr1221/1222) (rabbit) Cat. No. 2243, p-ERK1/2 (p44/42 MAP Kinase, Thr202/Tyr204) Antibody (rabbit) Cat. No. 9101, : t-EGFR-Antibody (rabbit) Cat. No. 2232, p-EGFR-Antibody (Tyr1068) (rabbit) Cat. No. 2234, and p-EGFR-Antibody (Tyr1086) (rabbit).

    Techniques: Western Blot, Labeling, Isolation

    Panel a) and c) are represenatative Western Blots following immunoprecipitations (IP) with either total-erbB2 or total-EGFR antibody and subsequent immunoblotting (IB) with both antibodies individually. Panel b) and d) represents the mean ratio of erbB2/EGFR dimers as assessed by densitometry for non-diabetic controls, (C), diabetic (D) and diabetic animals chronically treated with AG825 (+AG825) or AG1478 (+ AG1478) (both at dose of 1 mg/kg/ alt-diem ). N = 4; Asterisk (*) indicates significantly different (p<0.05) mean values from normal non-diabetic rats (C) whereas hash (#) indicates significantly different mean values (p<0.05) from diabetic rats (D).

    Journal: PLoS ONE

    Article Title: Activation of ErbB2 and Downstream Signalling via Rho Kinases and ERK1/2 Contributes to Diabetes-Induced Vascular Dysfunction

    doi: 10.1371/journal.pone.0067813

    Figure Lengend Snippet: Panel a) and c) are represenatative Western Blots following immunoprecipitations (IP) with either total-erbB2 or total-EGFR antibody and subsequent immunoblotting (IB) with both antibodies individually. Panel b) and d) represents the mean ratio of erbB2/EGFR dimers as assessed by densitometry for non-diabetic controls, (C), diabetic (D) and diabetic animals chronically treated with AG825 (+AG825) or AG1478 (+ AG1478) (both at dose of 1 mg/kg/ alt-diem ). N = 4; Asterisk (*) indicates significantly different (p<0.05) mean values from normal non-diabetic rats (C) whereas hash (#) indicates significantly different mean values (p<0.05) from diabetic rats (D).

    Article Snippet: The following antibodies from Cell Signaling (USA) were used in this study: t-Her2/ErbB2-Antibody (29D8) (rabbit) Cat. No. 2165, p-Her2/ErbB2-Antibody (Tyr877) (rabbit) Cat. No. 2241, p-Her2/ErbB2-Antibody (Tyr1248; labeled in figures as Y1248a) (rabbit) Cat. No. 2247, p-Her2/ErbB2-Antibody (Tyr1248/EGFR Tyr1173; labelled as Y1248b) (rabbit) Cat. No. 2244, p-Her2/ErbB2-Antibody (Tyr1221/1222) (rabbit) Cat. No. 2243, p-ERK1/2 (p44/42 MAP Kinase, Thr202/Tyr204) Antibody (rabbit) Cat. No. 9101, : t-EGFR-Antibody (rabbit) Cat. No. 2232, p-EGFR-Antibody (Tyr1068) (rabbit) Cat. No. 2234, and p-EGFR-Antibody (Tyr1086) (rabbit).

    Techniques: Western Blot

    A) Panel i) is a representative Western blot showing total (t-) or phosphorylated (p) levels of the stated proteins in VSMC grown in normal (5.5mM) D-glucose (NG), high glucose (25.5mM D-glucose; HG) or HG cotreated with increasing micromolar doses of AG825 (+ AG825). Panels ii-viii) are densitometry histograms showing total (t-) or phosphorylated (p-) levels of the stated proteins normalized to actin. B) Panel i) is a representative Western blot showing total (t-) or phosphorylated (p) levels of the stated proteins in VSMC grown in high glucose (25.5mM D-glucose; HG) or HG cotreated with increasing doses of anti-ErbB2 siRNA (ErbB2 siRNA) or non-targeting control siRNA (C siRNA). Panels ii-viii) are densitometry histograms showing total (t-) or phosphorylated (p-) levels of the stated proteins normalized to actin. N = 5; Mean±SD. Asterisk (*) indicates significantly different (p<0.05) mean values from normal non-diabetic rats (C) whereas hash (#) indicates significantly different mean values (p<0.05) from diabetic rats (D).

    Journal: PLoS ONE

    Article Title: Activation of ErbB2 and Downstream Signalling via Rho Kinases and ERK1/2 Contributes to Diabetes-Induced Vascular Dysfunction

    doi: 10.1371/journal.pone.0067813

    Figure Lengend Snippet: A) Panel i) is a representative Western blot showing total (t-) or phosphorylated (p) levels of the stated proteins in VSMC grown in normal (5.5mM) D-glucose (NG), high glucose (25.5mM D-glucose; HG) or HG cotreated with increasing micromolar doses of AG825 (+ AG825). Panels ii-viii) are densitometry histograms showing total (t-) or phosphorylated (p-) levels of the stated proteins normalized to actin. B) Panel i) is a representative Western blot showing total (t-) or phosphorylated (p) levels of the stated proteins in VSMC grown in high glucose (25.5mM D-glucose; HG) or HG cotreated with increasing doses of anti-ErbB2 siRNA (ErbB2 siRNA) or non-targeting control siRNA (C siRNA). Panels ii-viii) are densitometry histograms showing total (t-) or phosphorylated (p-) levels of the stated proteins normalized to actin. N = 5; Mean±SD. Asterisk (*) indicates significantly different (p<0.05) mean values from normal non-diabetic rats (C) whereas hash (#) indicates significantly different mean values (p<0.05) from diabetic rats (D).

    Article Snippet: The following antibodies from Cell Signaling (USA) were used in this study: t-Her2/ErbB2-Antibody (29D8) (rabbit) Cat. No. 2165, p-Her2/ErbB2-Antibody (Tyr877) (rabbit) Cat. No. 2241, p-Her2/ErbB2-Antibody (Tyr1248; labeled in figures as Y1248a) (rabbit) Cat. No. 2247, p-Her2/ErbB2-Antibody (Tyr1248/EGFR Tyr1173; labelled as Y1248b) (rabbit) Cat. No. 2244, p-Her2/ErbB2-Antibody (Tyr1221/1222) (rabbit) Cat. No. 2243, p-ERK1/2 (p44/42 MAP Kinase, Thr202/Tyr204) Antibody (rabbit) Cat. No. 9101, : t-EGFR-Antibody (rabbit) Cat. No. 2232, p-EGFR-Antibody (Tyr1068) (rabbit) Cat. No. 2234, and p-EGFR-Antibody (Tyr1086) (rabbit).

    Techniques: Western Blot