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  • 95
    Millipore her2 erbb2
    Her2 Erbb2, supplied by Millipore, used in various techniques. Bioz Stars score: 95/100, based on 13 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    Millipore erbb2 inhibitor
    Schwann cell Neuregulin-1 type 1 (NRG1-I) activates <t>ErbB2</t> and Mek/Erk signaling. a Phospho protein explorer antibody array with sciatic nerve protein extracts from 30-day-old mice. Heat map of the top 15 pathways that show inverse regulation between comparison one (NRG1cKO versus CMT1A, left lane) and two (CMT1A versus CMT1A-NRG1cKO, right lane), ranked by activation Z -score. Nrg1/ErbB2 and Erk/MAPK signaling (highlighted in magenta) were found among the top deregulated pathways, two technical replicates of n = 5 pooled samples per genotype were performed and data were analyzed by Ingenuity Pathway Analysis ( p value cutoff: log 45, see Methods for details). b Western blot analyses (left panels) and respective quantifications (right panels) of sciatic nerve lysates of 4-month-old mice showing increased expression and phosphorylation of ErbB2 (normalized to whole-protein-stained membrane, see Supplementary Fig. 5 ) and increased phosphorylation of Erk1/2 in CMT1A compared to wild-type mice. Both ErbB2 and Erk1/2 phosphorylation are normalized in CMT1A-NRG1cKO mice ( n = 3 per group, one-way analysis of variance (ANOVA) and Tukey’s post test; for full blots, see Supplementary Fig. 5 ). c Western blot analyses (left panels) and respective quantifications (right panels) of sciatic nerve lysates of 4-month-old mice showing hyperphosphorylation of ErbB2 (normalized to whole-protein-stained membrane, see Supplementary Figure 5 ) and Erk1/2 in NRG1-I OE mice compared to controls ( n = 3 per group, Student’s T test). Western blots of ErbB2 and pErbB2 were conducted in parallel with the same samples from the same protein preparations on two different membranes. d Western blot of primary CMT1A rat Schwann cells against phosphorylated and constitutive Erk1/2. Schwann cells were treated with or without ErbB2 inhibitor (10 µM, CAS 928207-02-7) for 6 h, which resulted in decreased phosphorylation of Erk1/2. Quantification of four independent experiments is shown (right panel, paired Student’s T test). e Relative mRNA expression of dedifferentiation and immaturity markers in sciatic nerve lysates (upper row: wild type n = 5, NRG1cKO n = 4, CMT1A n = 4, CMT1A-NRG1cKO n = 5, one-way ANOVA with Tukey’s post test; lower row: control n = 5, NRG1-I OE n = 4, Student’s T test). f Immunohistochemical analysis of the cJUN in sciatic nerve cross-sections from 4-month-old wild-type, CMT1A, CMT1A-NRG1cKO, and NRG1-I OE mice. Representative pictures are shown in the upper panels displaying cJUN expression (magenta, myelin MPZ in green) in myelinating (closed arrows) and supernumerary (open arrows) Schwann cells in CMT1A and NRG1-OE mice. Quantification of cJUN-positive nuclei from myelinating Schwann cells is shown in the panel below (control, n = 4; CMT1A, n = 3; CMT1A-NRG1cKO mice, n = 3; NRG1-I OE, n = 3; one-way ANOVA with Tukey’s post test). Source data are provided as a source data file. All respective p values are depicted as a range of significance with * p
    Erbb2 Inhibitor, supplied by Millipore, used in various techniques. Bioz Stars score: 91/100, based on 15 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore erbb2 ⓟ alexa488
    Schwann cell Neuregulin-1 type 1 (NRG1-I) activates <t>ErbB2</t> and Mek/Erk signaling. a Phospho protein explorer antibody array with sciatic nerve protein extracts from 30-day-old mice. Heat map of the top 15 pathways that show inverse regulation between comparison one (NRG1cKO versus CMT1A, left lane) and two (CMT1A versus CMT1A-NRG1cKO, right lane), ranked by activation Z -score. Nrg1/ErbB2 and Erk/MAPK signaling (highlighted in magenta) were found among the top deregulated pathways, two technical replicates of n = 5 pooled samples per genotype were performed and data were analyzed by Ingenuity Pathway Analysis ( p value cutoff: log 45, see Methods for details). b Western blot analyses (left panels) and respective quantifications (right panels) of sciatic nerve lysates of 4-month-old mice showing increased expression and phosphorylation of ErbB2 (normalized to whole-protein-stained membrane, see Supplementary Fig. 5 ) and increased phosphorylation of Erk1/2 in CMT1A compared to wild-type mice. Both ErbB2 and Erk1/2 phosphorylation are normalized in CMT1A-NRG1cKO mice ( n = 3 per group, one-way analysis of variance (ANOVA) and Tukey’s post test; for full blots, see Supplementary Fig. 5 ). c Western blot analyses (left panels) and respective quantifications (right panels) of sciatic nerve lysates of 4-month-old mice showing hyperphosphorylation of ErbB2 (normalized to whole-protein-stained membrane, see Supplementary Figure 5 ) and Erk1/2 in NRG1-I OE mice compared to controls ( n = 3 per group, Student’s T test). Western blots of ErbB2 and pErbB2 were conducted in parallel with the same samples from the same protein preparations on two different membranes. d Western blot of primary CMT1A rat Schwann cells against phosphorylated and constitutive Erk1/2. Schwann cells were treated with or without ErbB2 inhibitor (10 µM, CAS 928207-02-7) for 6 h, which resulted in decreased phosphorylation of Erk1/2. Quantification of four independent experiments is shown (right panel, paired Student’s T test). e Relative mRNA expression of dedifferentiation and immaturity markers in sciatic nerve lysates (upper row: wild type n = 5, NRG1cKO n = 4, CMT1A n = 4, CMT1A-NRG1cKO n = 5, one-way ANOVA with Tukey’s post test; lower row: control n = 5, NRG1-I OE n = 4, Student’s T test). f Immunohistochemical analysis of the cJUN in sciatic nerve cross-sections from 4-month-old wild-type, CMT1A, CMT1A-NRG1cKO, and NRG1-I OE mice. Representative pictures are shown in the upper panels displaying cJUN expression (magenta, myelin MPZ in green) in myelinating (closed arrows) and supernumerary (open arrows) Schwann cells in CMT1A and NRG1-OE mice. Quantification of cJUN-positive nuclei from myelinating Schwann cells is shown in the panel below (control, n = 4; CMT1A, n = 3; CMT1A-NRG1cKO mice, n = 3; NRG1-I OE, n = 3; one-way ANOVA with Tukey’s post test). Source data are provided as a source data file. All respective p values are depicted as a range of significance with * p
    Erbb2 ⓟ Alexa488, supplied by Millipore, used in various techniques. Bioz Stars score: 81/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore α c erbb2
    MM-121 in combination with trastuzumab significantly inhibits in vivo growth of tumor xenografts established from BT474-HR20 trastuzumab-resistant breast cancer cells. BT474-HR20 cells were s.c. injected into nude mice to establish tumor xenografts. The tumor-bearing mice (n = 5) received i.p. injections of PBS, trastuzumab, MM-121, or trastuzumab plus MM-121 as described in the Materials and Methods. After 6 treatments, the mice were euthanized at day 36 post injection of tumor cells, and all tumors were excised for histology and IHC analysis. A , The graphs show the tumor growth curves. Bars , SD. The combinations of MM-121 and trastuzumab significantly inhibited tumor growth as compared to control or single Ab treatment. B , Data show the representative tumors with hematoxylin and eosin (H E) staining and IHC analysis of <t>erbB2</t> and erbB3. The residual tumor cells obtained from combinatorial treatments retained similar expression levels of erbB2/erbB3 receptors on the cell membrane.
    α C Erbb2, supplied by Millipore, used in various techniques. Bioz Stars score: 81/100, based on 77 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore erbb2 inhibitor ag1478
    Regulation of MYCN expression by BMP and EGF signaling pathways. A , Wild type E11.5 embryonic hearts were cultured 2 hours with BMP10 (100 ng/ml), BMP inhibitor DMH1 (8 uM), Neuregulin-1 (625 ng/ml), or <t>ERBB2</t> inhibitor <t>AG1478</t> (100 uM). Western analysis
    Erbb2 Inhibitor Ag1478, supplied by Millipore, used in various techniques. Bioz Stars score: 77/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    83
    Millipore phosphorylated erbb2 tyr1248
    Regulation of MYCN expression by BMP and EGF signaling pathways. A , Wild type E11.5 embryonic hearts were cultured 2 hours with BMP10 (100 ng/ml), BMP inhibitor DMH1 (8 uM), Neuregulin-1 (625 ng/ml), or <t>ERBB2</t> inhibitor <t>AG1478</t> (100 uM). Western analysis
    Phosphorylated Erbb2 Tyr1248, supplied by Millipore, used in various techniques. Bioz Stars score: 83/100, based on 9 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    76
    Millipore her 2 neu elisa kits
    Relationship between Ets-1 52 kDa protein and <t>HER-2/</t> <t>neu</t> (fmol mg −1 ). Ets-1 protein measurements were carried out using Western blotting. Arbitrary units were assigned to each protein band following scanning densitometry. Values are expressed as a ratio to β -actin. HER-2/ neu levels were measured using <t>ELISA.</t> Data was analysed using the nonparametric Spearman rank test.
    Her 2 Neu Elisa Kits, supplied by Millipore, used in various techniques. Bioz Stars score: 76/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore erbb2 ab3
    Relationship between Ets-1 52 kDa protein and <t>HER-2/</t> <t>neu</t> (fmol mg −1 ). Ets-1 protein measurements were carried out using Western blotting. Arbitrary units were assigned to each protein band following scanning densitometry. Values are expressed as a ratio to β -actin. HER-2/ neu levels were measured using <t>ELISA.</t> Data was analysed using the nonparametric Spearman rank test.
    Erbb2 Ab3, supplied by Millipore, used in various techniques. Bioz Stars score: 77/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore p erbb2 tyr 1248
    Relationship between Ets-1 52 kDa protein and <t>HER-2/</t> <t>neu</t> (fmol mg −1 ). Ets-1 protein measurements were carried out using Western blotting. Arbitrary units were assigned to each protein band following scanning densitometry. Values are expressed as a ratio to β -actin. HER-2/ neu levels were measured using <t>ELISA.</t> Data was analysed using the nonparametric Spearman rank test.
    P Erbb2 Tyr 1248, supplied by Millipore, used in various techniques. Bioz Stars score: 80/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore erbb2 receptor inhibitor ag825
    A : microRNA analysis of mmu-miR-296–3p, mmu-miR-200c*, and mmu-miR-465b-5p was differentially regulated by NRG1 and ErbB receptor inhibition. Cardiac differentiation of ESCs was performed by the hanging drop method. Cells were incubated with NRG1, <t>ErbB2</t> inhibitor <t>AG825</t> (1 μM), or ErbB1/ErbB2/ErbB4 inhibitor (1 nM) during day 5–7 . Cells were then incubated without stimulation. RNA was collected on day 9 . Accession number and name of the microRNAs are shown. B : mmu-miR-296–3p, mmu-miR-200c*, and mmu-miR-465b-5p were differentially expressed during hanging drop-induced mesoderm formation of ESCs. The expression of mmu-miR-296–3p and mmu-miR-200c* was increased during hanging drop-induced ESC differentiation. Expression of mmu-miR- 465b-5p was decreased during hanging drop-induced ESC differentiation. ESC differentiation was induced by the hanging drop method. RNA was collected on days 0 , 3, 5 , and 9 . Expression of mmu-miR-296–3p, mmu-miR-200c*, and mmu-miR-465b-5p was assessed by real-time PCR. Data were normalized to snoRNA202 and are presented as fold expression relative to the mRNA level of undifferentiated ESCs ( day 0 ). The results are from three independent experiments. * P
    Erbb2 Receptor Inhibitor Ag825, supplied by Millipore, used in various techniques. Bioz Stars score: 76/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore erbb2 inhibitor pd 168393
    A : microRNA analysis of mmu-miR-296–3p, mmu-miR-200c*, and mmu-miR-465b-5p was differentially regulated by NRG1 and ErbB receptor inhibition. Cardiac differentiation of ESCs was performed by the hanging drop method. Cells were incubated with NRG1, <t>ErbB2</t> inhibitor <t>AG825</t> (1 μM), or ErbB1/ErbB2/ErbB4 inhibitor (1 nM) during day 5–7 . Cells were then incubated without stimulation. RNA was collected on day 9 . Accession number and name of the microRNAs are shown. B : mmu-miR-296–3p, mmu-miR-200c*, and mmu-miR-465b-5p were differentially expressed during hanging drop-induced mesoderm formation of ESCs. The expression of mmu-miR-296–3p and mmu-miR-200c* was increased during hanging drop-induced ESC differentiation. Expression of mmu-miR- 465b-5p was decreased during hanging drop-induced ESC differentiation. ESC differentiation was induced by the hanging drop method. RNA was collected on days 0 , 3, 5 , and 9 . Expression of mmu-miR-296–3p, mmu-miR-200c*, and mmu-miR-465b-5p was assessed by real-time PCR. Data were normalized to snoRNA202 and are presented as fold expression relative to the mRNA level of undifferentiated ESCs ( day 0 ). The results are from three independent experiments. * P
    Erbb2 Inhibitor Pd 168393, supplied by Millipore, used in various techniques. Bioz Stars score: 77/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore anti p1248y erbb2
    A : microRNA analysis of mmu-miR-296–3p, mmu-miR-200c*, and mmu-miR-465b-5p was differentially regulated by NRG1 and ErbB receptor inhibition. Cardiac differentiation of ESCs was performed by the hanging drop method. Cells were incubated with NRG1, <t>ErbB2</t> inhibitor <t>AG825</t> (1 μM), or ErbB1/ErbB2/ErbB4 inhibitor (1 nM) during day 5–7 . Cells were then incubated without stimulation. RNA was collected on day 9 . Accession number and name of the microRNAs are shown. B : mmu-miR-296–3p, mmu-miR-200c*, and mmu-miR-465b-5p were differentially expressed during hanging drop-induced mesoderm formation of ESCs. The expression of mmu-miR-296–3p and mmu-miR-200c* was increased during hanging drop-induced ESC differentiation. Expression of mmu-miR- 465b-5p was decreased during hanging drop-induced ESC differentiation. ESC differentiation was induced by the hanging drop method. RNA was collected on days 0 , 3, 5 , and 9 . Expression of mmu-miR-296–3p, mmu-miR-200c*, and mmu-miR-465b-5p was assessed by real-time PCR. Data were normalized to snoRNA202 and are presented as fold expression relative to the mRNA level of undifferentiated ESCs ( day 0 ). The results are from three independent experiments. * P
    Anti P1248y Erbb2, supplied by Millipore, used in various techniques. Bioz Stars score: 87/100, based on 12 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore erbb2 y1248p
    Reverse phase protein analyses of <t>ErbB2</t> + Ph + ALL with canertinib treatment. (A) Unsupervised clustering analyses were performed on RPPA data from Z181 or Z119 cells treated with 0.1–5 µM of canertinib for 18 hours. To generate heat maps, total protein and phosphoprotein levels were quantified by RPPA, and data were centered on the mean. Intensifying red color indicates increasing protein or phosphoprotein expression relative to the mean, black indicates the mean value, and intensifying green color indicates decreasing levels. (B) Supervised clustering showing relative changes in expression of pro-apoptotic proteins (Bim, cleaved PARP), protein kinase Cs, heat shock proteins, p38 mitogen activated protein kinase, and p38 T180/Y182p; five components of the phosphatidylinositol 3 kinase signaling pathway (Akt T308p, Akt S473p, p70S6-kinase T389p, S6-kinase S235/36p, and S6-kinase S240/44p); and ErbB2 <t>Y1248p</t> in the two cell lines after treatment.
    Erbb2 Y1248p, supplied by Millipore, used in various techniques. Bioz Stars score: 78/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore p erbb2
    Reverse phase protein analyses of <t>ErbB2</t> + Ph + ALL with canertinib treatment. (A) Unsupervised clustering analyses were performed on RPPA data from Z181 or Z119 cells treated with 0.1–5 µM of canertinib for 18 hours. To generate heat maps, total protein and phosphoprotein levels were quantified by RPPA, and data were centered on the mean. Intensifying red color indicates increasing protein or phosphoprotein expression relative to the mean, black indicates the mean value, and intensifying green color indicates decreasing levels. (B) Supervised clustering showing relative changes in expression of pro-apoptotic proteins (Bim, cleaved PARP), protein kinase Cs, heat shock proteins, p38 mitogen activated protein kinase, and p38 T180/Y182p; five components of the phosphatidylinositol 3 kinase signaling pathway (Akt T308p, Akt S473p, p70S6-kinase T389p, S6-kinase S235/36p, and S6-kinase S240/44p); and ErbB2 <t>Y1248p</t> in the two cell lines after treatment.
    P Erbb2, supplied by Millipore, used in various techniques. Bioz Stars score: 83/100, based on 8 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore t erbb2
    Reverse phase protein analyses of <t>ErbB2</t> + Ph + ALL with canertinib treatment. (A) Unsupervised clustering analyses were performed on RPPA data from Z181 or Z119 cells treated with 0.1–5 µM of canertinib for 18 hours. To generate heat maps, total protein and phosphoprotein levels were quantified by RPPA, and data were centered on the mean. Intensifying red color indicates increasing protein or phosphoprotein expression relative to the mean, black indicates the mean value, and intensifying green color indicates decreasing levels. (B) Supervised clustering showing relative changes in expression of pro-apoptotic proteins (Bim, cleaved PARP), protein kinase Cs, heat shock proteins, p38 mitogen activated protein kinase, and p38 T180/Y182p; five components of the phosphatidylinositol 3 kinase signaling pathway (Akt T308p, Akt S473p, p70S6-kinase T389p, S6-kinase S235/36p, and S6-kinase S240/44p); and ErbB2 <t>Y1248p</t> in the two cell lines after treatment.
    T Erbb2, supplied by Millipore, used in various techniques. Bioz Stars score: 82/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore rabbit anti erbb2
    Msi1 'knockdown' by a shRNA preferentially reduces CD133 + cells and stem cell marker expression . A . Lentivirus expression of an Msi1 shRNA. Left, 'Knockdown' (KD) of Msi1 in MCF-7 and T47D spheroid cells by an Msi1 shRNA (Msi1 shRNA) reduces Msi1, CD133, <t>ErbB2</t> and pERK expression vs. the control shRNA (Contr shRNA). Right panel, quantitation of the western blot indicates that Msi1 was reduced by 77-84% in MCF-7 and T47D cells. B . Msi1 KD reduces stem cell marker expression in MCF-7 and T47D cells. CD133, Nanog, Oct4, Sox2, c-Myc and Bmi1 mRNA levels were determined by qRT-PCR. All stem cell markers, with the exception of Oct4 in T47D cells, were reduced by Msi1 knockdown. C . Left panel, Msi1 KD reduces Notch1 and increases p21 CIP1 in MCF-7 and T47D spheroid cells. IC, Notch intracellular domain; FL, full-length Notch1. Middle panel, quantitation of Notch1 expression. Right panel, quantitation of p21 CIP1 expression. D . Msi1 KD reduces Notch1 mRNA 2.7-5-fold, but not p21 CIP1 , in MCF-7 and T47D spheroid cells, respectively.
    Rabbit Anti Erbb2, supplied by Millipore, used in various techniques. Bioz Stars score: 89/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore rabbit anti her 2 neu antibody
    IbTX differentially regulates oncogenic pathways. (A) IbTX attenuated the <t>HER-2/neu</t> levels in the UACC893 cells ( HER-2/neu , UACC893 ). SK-BR-3 cells express full length ( HER-2/neu , SK-BR-3 ) and truncated ( p95 HER-2/neu , SK-BR-3 ) oncoprotein isoforms with both being increased by IbTX. (B) IbTX downregulated the full length ( β-catenin ) but not truncated ( trβ-catenin ) β-catenin isoforms in the UACC893 and MDA-MB-231 cells. (C) In the UACC893 cells, IbTX attenuated phosphorylated ( T308pAkt , UACC893 ) but not total Akt ( Akt , UACC893 ). Both phosphorylated ( T308pAkt , MDA-MB-231 ) and total ( Akt , MDA-MB-231 ) Akt were decreased in the MDA-MB-231 cells with IbTX. In SK-BR-3 cells, phosphorylated ( T308pAkt , SK-BR-3 ) and total ( Akt , SK-BR-3 ) Akt increased at high IbTX concentrations. (D) GAPDH was used in the UACC893, MDA-MB-231 and SK-BR-3 cell immunoblotting to ensure equal protein loading. IbTX, iberiotoxin.
    Rabbit Anti Her 2 Neu Antibody, supplied by Millipore, used in various techniques. Bioz Stars score: 79/100, based on 8 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore phospho erbb 2 her 2 tyr1248
    IbTX differentially regulates oncogenic pathways. (A) IbTX attenuated the <t>HER-2/neu</t> levels in the UACC893 cells ( HER-2/neu , UACC893 ). SK-BR-3 cells express full length ( HER-2/neu , SK-BR-3 ) and truncated ( p95 HER-2/neu , SK-BR-3 ) oncoprotein isoforms with both being increased by IbTX. (B) IbTX downregulated the full length ( β-catenin ) but not truncated ( trβ-catenin ) β-catenin isoforms in the UACC893 and MDA-MB-231 cells. (C) In the UACC893 cells, IbTX attenuated phosphorylated ( T308pAkt , UACC893 ) but not total Akt ( Akt , UACC893 ). Both phosphorylated ( T308pAkt , MDA-MB-231 ) and total ( Akt , MDA-MB-231 ) Akt were decreased in the MDA-MB-231 cells with IbTX. In SK-BR-3 cells, phosphorylated ( T308pAkt , SK-BR-3 ) and total ( Akt , SK-BR-3 ) Akt increased at high IbTX concentrations. (D) GAPDH was used in the UACC893, MDA-MB-231 and SK-BR-3 cell immunoblotting to ensure equal protein loading. IbTX, iberiotoxin.
    Phospho Erbb 2 Her 2 Tyr1248, supplied by Millipore, used in various techniques. Bioz Stars score: 78/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    IbTX differentially regulates oncogenic pathways. (A) IbTX attenuated the <t>HER-2/neu</t> levels in the UACC893 cells ( HER-2/neu , UACC893 ). SK-BR-3 cells express full length ( HER-2/neu , SK-BR-3 ) and truncated ( p95 HER-2/neu , SK-BR-3 ) oncoprotein isoforms with both being increased by IbTX. (B) IbTX downregulated the full length ( β-catenin ) but not truncated ( trβ-catenin ) β-catenin isoforms in the UACC893 and MDA-MB-231 cells. (C) In the UACC893 cells, IbTX attenuated phosphorylated ( T308pAkt , UACC893 ) but not total Akt ( Akt , UACC893 ). Both phosphorylated ( T308pAkt , MDA-MB-231 ) and total ( Akt , MDA-MB-231 ) Akt were decreased in the MDA-MB-231 cells with IbTX. In SK-BR-3 cells, phosphorylated ( T308pAkt , SK-BR-3 ) and total ( Akt , SK-BR-3 ) Akt increased at high IbTX concentrations. (D) GAPDH was used in the UACC893, MDA-MB-231 and SK-BR-3 cell immunoblotting to ensure equal protein loading. IbTX, iberiotoxin.
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    Image Search Results


    Schwann cell Neuregulin-1 type 1 (NRG1-I) activates ErbB2 and Mek/Erk signaling. a Phospho protein explorer antibody array with sciatic nerve protein extracts from 30-day-old mice. Heat map of the top 15 pathways that show inverse regulation between comparison one (NRG1cKO versus CMT1A, left lane) and two (CMT1A versus CMT1A-NRG1cKO, right lane), ranked by activation Z -score. Nrg1/ErbB2 and Erk/MAPK signaling (highlighted in magenta) were found among the top deregulated pathways, two technical replicates of n = 5 pooled samples per genotype were performed and data were analyzed by Ingenuity Pathway Analysis ( p value cutoff: log 45, see Methods for details). b Western blot analyses (left panels) and respective quantifications (right panels) of sciatic nerve lysates of 4-month-old mice showing increased expression and phosphorylation of ErbB2 (normalized to whole-protein-stained membrane, see Supplementary Fig. 5 ) and increased phosphorylation of Erk1/2 in CMT1A compared to wild-type mice. Both ErbB2 and Erk1/2 phosphorylation are normalized in CMT1A-NRG1cKO mice ( n = 3 per group, one-way analysis of variance (ANOVA) and Tukey’s post test; for full blots, see Supplementary Fig. 5 ). c Western blot analyses (left panels) and respective quantifications (right panels) of sciatic nerve lysates of 4-month-old mice showing hyperphosphorylation of ErbB2 (normalized to whole-protein-stained membrane, see Supplementary Figure 5 ) and Erk1/2 in NRG1-I OE mice compared to controls ( n = 3 per group, Student’s T test). Western blots of ErbB2 and pErbB2 were conducted in parallel with the same samples from the same protein preparations on two different membranes. d Western blot of primary CMT1A rat Schwann cells against phosphorylated and constitutive Erk1/2. Schwann cells were treated with or without ErbB2 inhibitor (10 µM, CAS 928207-02-7) for 6 h, which resulted in decreased phosphorylation of Erk1/2. Quantification of four independent experiments is shown (right panel, paired Student’s T test). e Relative mRNA expression of dedifferentiation and immaturity markers in sciatic nerve lysates (upper row: wild type n = 5, NRG1cKO n = 4, CMT1A n = 4, CMT1A-NRG1cKO n = 5, one-way ANOVA with Tukey’s post test; lower row: control n = 5, NRG1-I OE n = 4, Student’s T test). f Immunohistochemical analysis of the cJUN in sciatic nerve cross-sections from 4-month-old wild-type, CMT1A, CMT1A-NRG1cKO, and NRG1-I OE mice. Representative pictures are shown in the upper panels displaying cJUN expression (magenta, myelin MPZ in green) in myelinating (closed arrows) and supernumerary (open arrows) Schwann cells in CMT1A and NRG1-OE mice. Quantification of cJUN-positive nuclei from myelinating Schwann cells is shown in the panel below (control, n = 4; CMT1A, n = 3; CMT1A-NRG1cKO mice, n = 3; NRG1-I OE, n = 3; one-way ANOVA with Tukey’s post test). Source data are provided as a source data file. All respective p values are depicted as a range of significance with * p

    Journal: Nature Communications

    Article Title: NRG1 type I dependent autoparacrine stimulation of Schwann cells in onion bulbs of peripheral neuropathies

    doi: 10.1038/s41467-019-09385-6

    Figure Lengend Snippet: Schwann cell Neuregulin-1 type 1 (NRG1-I) activates ErbB2 and Mek/Erk signaling. a Phospho protein explorer antibody array with sciatic nerve protein extracts from 30-day-old mice. Heat map of the top 15 pathways that show inverse regulation between comparison one (NRG1cKO versus CMT1A, left lane) and two (CMT1A versus CMT1A-NRG1cKO, right lane), ranked by activation Z -score. Nrg1/ErbB2 and Erk/MAPK signaling (highlighted in magenta) were found among the top deregulated pathways, two technical replicates of n = 5 pooled samples per genotype were performed and data were analyzed by Ingenuity Pathway Analysis ( p value cutoff: log 45, see Methods for details). b Western blot analyses (left panels) and respective quantifications (right panels) of sciatic nerve lysates of 4-month-old mice showing increased expression and phosphorylation of ErbB2 (normalized to whole-protein-stained membrane, see Supplementary Fig. 5 ) and increased phosphorylation of Erk1/2 in CMT1A compared to wild-type mice. Both ErbB2 and Erk1/2 phosphorylation are normalized in CMT1A-NRG1cKO mice ( n = 3 per group, one-way analysis of variance (ANOVA) and Tukey’s post test; for full blots, see Supplementary Fig. 5 ). c Western blot analyses (left panels) and respective quantifications (right panels) of sciatic nerve lysates of 4-month-old mice showing hyperphosphorylation of ErbB2 (normalized to whole-protein-stained membrane, see Supplementary Figure 5 ) and Erk1/2 in NRG1-I OE mice compared to controls ( n = 3 per group, Student’s T test). Western blots of ErbB2 and pErbB2 were conducted in parallel with the same samples from the same protein preparations on two different membranes. d Western blot of primary CMT1A rat Schwann cells against phosphorylated and constitutive Erk1/2. Schwann cells were treated with or without ErbB2 inhibitor (10 µM, CAS 928207-02-7) for 6 h, which resulted in decreased phosphorylation of Erk1/2. Quantification of four independent experiments is shown (right panel, paired Student’s T test). e Relative mRNA expression of dedifferentiation and immaturity markers in sciatic nerve lysates (upper row: wild type n = 5, NRG1cKO n = 4, CMT1A n = 4, CMT1A-NRG1cKO n = 5, one-way ANOVA with Tukey’s post test; lower row: control n = 5, NRG1-I OE n = 4, Student’s T test). f Immunohistochemical analysis of the cJUN in sciatic nerve cross-sections from 4-month-old wild-type, CMT1A, CMT1A-NRG1cKO, and NRG1-I OE mice. Representative pictures are shown in the upper panels displaying cJUN expression (magenta, myelin MPZ in green) in myelinating (closed arrows) and supernumerary (open arrows) Schwann cells in CMT1A and NRG1-OE mice. Quantification of cJUN-positive nuclei from myelinating Schwann cells is shown in the panel below (control, n = 4; CMT1A, n = 3; CMT1A-NRG1cKO mice, n = 3; NRG1-I OE, n = 3; one-way ANOVA with Tukey’s post test). Source data are provided as a source data file. All respective p values are depicted as a range of significance with * p

    Article Snippet: Rat wild-type Schwann cells were treated with an ErbB2-Inhibitor (CAS 928207-02-7, Calbiochem, 30 µM in dimethyl sulfoxide (DMSO)) or DMSO as control (1:1000).

    Techniques: Ab Array, Mouse Assay, Activation Assay, Western Blot, Expressing, Staining, Immunohistochemistry

    Schwann cell Nrg1 promotes survival and migration of Schwann cells. a Survival assay with primary rat Schwann cells after 2 days in vitro (2div) with or without an ErbB2 inhibitor (CAS 928207-02-7, 30 µM). Representative before/after pictures (left panels) with quantification (right panel; a – c : n = 3 independent Schwann cell preparations per group, paired Student’s T test). b Survival assay with primary mouse Schwann cells from wild-type and NRG1cKO mice after 3div. c In the same experiment as in b , the apoptosis rate was assessed by terminal deoxinucleotidyl transferase-mediated dUTP-fluorescein nick end labeling (TUNEL) assay. d Quantification of Schwann cell apoptosis as measured by percentage of TUNEL-positive nuclei on sciatic nerve longitudinal sections from 11-day-old mice (wild type n = 6, NRG1cKO n = 5, CMT1A n = 6, CMT1A-NRG1cKO n = 9, NRG1-I OE n = 3, one-way analysis of variance (ANOVA), Tukey’s post test). e Quantification of Schwann cell nuclei per area (15,740 µm 2 ) on electron micrographs of 4-month-old wild type ( n = 4), NRG1cKO ( n = 6), CMT1A ( n = 6), and CMT1A-NRG1cKO ( n = 6) mice (one-way ANOVA and Tukey’s post test). f Fluorescent immunohistochemistry on tibial nerve cross-sections from 4-month-old control ( n = 4) and Neuregulin-1 type 1 (NRG1-I) overexpression (OE) ( n = 3) mice against SOX10 (left panels) and respective quantification (right panel, Student’s T test). g Schematic set-up of Boyden chamber migration experiments (left panel) and respective quantification (right panel) of the percentage of cells that migrated from the top compartment through the mesh (green in left panel). The top compartment was always seeded with wild-type (wt) Schwann cells, whereas in the bottom compartment either with wild type (wt, black, set to 100%) or NRG1cKO Schwann cells were seeded and the latter without (ko, red) or with (ko+ NRG1, blue) recombinant NRG1 (quantification of three independent experiments, one-way ANOVA, Tukey’s post test). h BaseScope TM in situ hybridization with a Nrg1-I probe (magenta) on sciatic nerve cross-sections of 18-day-old wild-type and CMT1A mice. Myelin was counterstained with MBP (green) and together with the phase contrast demonstrates Nrg1-I expression in myelinating Schwann cells in the CMT1A mouse (arrows, scale bar 5 µm). i BaseScope in situ hybridization with a Nrg1-I probe (magenta) on sciatic nerve cross-sections of a 4-month-old NRG1-OE mouse showing expression of Nrg1-I (magenta) in Schwann cells (S100, green). Note that supernumerary Schwann cells (closed arrows) are located nearby a Nrg1-I -expressing myelinating Schwann cell (open arrow, scale bar 5 µm, DAPI: nuclei). Source data are provided as a source data file. All respective p values are depicted as a range of significance with * p

    Journal: Nature Communications

    Article Title: NRG1 type I dependent autoparacrine stimulation of Schwann cells in onion bulbs of peripheral neuropathies

    doi: 10.1038/s41467-019-09385-6

    Figure Lengend Snippet: Schwann cell Nrg1 promotes survival and migration of Schwann cells. a Survival assay with primary rat Schwann cells after 2 days in vitro (2div) with or without an ErbB2 inhibitor (CAS 928207-02-7, 30 µM). Representative before/after pictures (left panels) with quantification (right panel; a – c : n = 3 independent Schwann cell preparations per group, paired Student’s T test). b Survival assay with primary mouse Schwann cells from wild-type and NRG1cKO mice after 3div. c In the same experiment as in b , the apoptosis rate was assessed by terminal deoxinucleotidyl transferase-mediated dUTP-fluorescein nick end labeling (TUNEL) assay. d Quantification of Schwann cell apoptosis as measured by percentage of TUNEL-positive nuclei on sciatic nerve longitudinal sections from 11-day-old mice (wild type n = 6, NRG1cKO n = 5, CMT1A n = 6, CMT1A-NRG1cKO n = 9, NRG1-I OE n = 3, one-way analysis of variance (ANOVA), Tukey’s post test). e Quantification of Schwann cell nuclei per area (15,740 µm 2 ) on electron micrographs of 4-month-old wild type ( n = 4), NRG1cKO ( n = 6), CMT1A ( n = 6), and CMT1A-NRG1cKO ( n = 6) mice (one-way ANOVA and Tukey’s post test). f Fluorescent immunohistochemistry on tibial nerve cross-sections from 4-month-old control ( n = 4) and Neuregulin-1 type 1 (NRG1-I) overexpression (OE) ( n = 3) mice against SOX10 (left panels) and respective quantification (right panel, Student’s T test). g Schematic set-up of Boyden chamber migration experiments (left panel) and respective quantification (right panel) of the percentage of cells that migrated from the top compartment through the mesh (green in left panel). The top compartment was always seeded with wild-type (wt) Schwann cells, whereas in the bottom compartment either with wild type (wt, black, set to 100%) or NRG1cKO Schwann cells were seeded and the latter without (ko, red) or with (ko+ NRG1, blue) recombinant NRG1 (quantification of three independent experiments, one-way ANOVA, Tukey’s post test). h BaseScope TM in situ hybridization with a Nrg1-I probe (magenta) on sciatic nerve cross-sections of 18-day-old wild-type and CMT1A mice. Myelin was counterstained with MBP (green) and together with the phase contrast demonstrates Nrg1-I expression in myelinating Schwann cells in the CMT1A mouse (arrows, scale bar 5 µm). i BaseScope in situ hybridization with a Nrg1-I probe (magenta) on sciatic nerve cross-sections of a 4-month-old NRG1-OE mouse showing expression of Nrg1-I (magenta) in Schwann cells (S100, green). Note that supernumerary Schwann cells (closed arrows) are located nearby a Nrg1-I -expressing myelinating Schwann cell (open arrow, scale bar 5 µm, DAPI: nuclei). Source data are provided as a source data file. All respective p values are depicted as a range of significance with * p

    Article Snippet: Rat wild-type Schwann cells were treated with an ErbB2-Inhibitor (CAS 928207-02-7, Calbiochem, 30 µM in dimethyl sulfoxide (DMSO)) or DMSO as control (1:1000).

    Techniques: Migration, Clonogenic Cell Survival Assay, In Vitro, Mouse Assay, End Labeling, TUNEL Assay, Immunohistochemistry, Over Expression, Recombinant, In Situ Hybridization, Expressing

    MM-121 in combination with trastuzumab significantly inhibits in vivo growth of tumor xenografts established from BT474-HR20 trastuzumab-resistant breast cancer cells. BT474-HR20 cells were s.c. injected into nude mice to establish tumor xenografts. The tumor-bearing mice (n = 5) received i.p. injections of PBS, trastuzumab, MM-121, or trastuzumab plus MM-121 as described in the Materials and Methods. After 6 treatments, the mice were euthanized at day 36 post injection of tumor cells, and all tumors were excised for histology and IHC analysis. A , The graphs show the tumor growth curves. Bars , SD. The combinations of MM-121 and trastuzumab significantly inhibited tumor growth as compared to control or single Ab treatment. B , Data show the representative tumors with hematoxylin and eosin (H E) staining and IHC analysis of erbB2 and erbB3. The residual tumor cells obtained from combinatorial treatments retained similar expression levels of erbB2/erbB3 receptors on the cell membrane.

    Journal: Molecular Cancer

    Article Title: The anti-erbB3 antibody MM-121/SAR256212 in combination with trastuzumab exerts potent antitumor activity against trastuzumab-resistant breast cancer cells

    doi: 10.1186/1476-4598-12-134

    Figure Lengend Snippet: MM-121 in combination with trastuzumab significantly inhibits in vivo growth of tumor xenografts established from BT474-HR20 trastuzumab-resistant breast cancer cells. BT474-HR20 cells were s.c. injected into nude mice to establish tumor xenografts. The tumor-bearing mice (n = 5) received i.p. injections of PBS, trastuzumab, MM-121, or trastuzumab plus MM-121 as described in the Materials and Methods. After 6 treatments, the mice were euthanized at day 36 post injection of tumor cells, and all tumors were excised for histology and IHC analysis. A , The graphs show the tumor growth curves. Bars , SD. The combinations of MM-121 and trastuzumab significantly inhibited tumor growth as compared to control or single Ab treatment. B , Data show the representative tumors with hematoxylin and eosin (H E) staining and IHC analysis of erbB2 and erbB3. The residual tumor cells obtained from combinatorial treatments retained similar expression levels of erbB2/erbB3 receptors on the cell membrane.

    Article Snippet: Immunohistochemistry Five micron thick paraffin sections were deparaffinized, antigens unmasked and immunohistochemically stained for Ki67 (Thermo Fisher Scientific; rabbit monoclonal SP6; cat# RM-9106-SO; dilution 1:500 in TBST + 1% BSA w/v), cleaved Caspase-3 (Cell Signaling Technology; rabbit polyclonal; cat#: 9661, 1:1000 in TBST + 1% BSA w/v), erbB2 (EMD Chemicals; mouse monoclonal 96G; cat#OP14T; dilution 1:500 in TBST + 1% BSA w/v), and erbB3 (Spring Bioscience, Pleasanton, CA; rabbit monoclonal SP71; cat# M3710; dilution 1:200 in TBST + 1% BSA w/v).

    Techniques: In Vivo, Injection, Mouse Assay, Immunohistochemistry, Staining, Expressing

    The addition of MM-121 enhances trastuzumab-induced cell cycle G1 arrest in erbB2+ breast cancer cell lines. SKBR3 and BT474 cells were untreated or treated with either trastuzumab or MM-121 alone, or their combinations for 24 hrs. A , Half of the cells were collected and subjected to western blot analyses with specific antibodies directed against E2F-1, Cyclin D1, p27 kip1 , or β-actin. The densitometry analyses of E2F-1, Cyclin D1, and p27 kip1 signals were shown underneath, and the arbitrary numbers indicate the intensities of each sample relative to controls, defined as 1.0. B C , The other half of the cells were collected for analysis of cell cycle distributions by flow cytometry as described in the Materials and Methods. Data show a representative of three independent experiments.

    Journal: Molecular Cancer

    Article Title: The anti-erbB3 antibody MM-121/SAR256212 in combination with trastuzumab exerts potent antitumor activity against trastuzumab-resistant breast cancer cells

    doi: 10.1186/1476-4598-12-134

    Figure Lengend Snippet: The addition of MM-121 enhances trastuzumab-induced cell cycle G1 arrest in erbB2+ breast cancer cell lines. SKBR3 and BT474 cells were untreated or treated with either trastuzumab or MM-121 alone, or their combinations for 24 hrs. A , Half of the cells were collected and subjected to western blot analyses with specific antibodies directed against E2F-1, Cyclin D1, p27 kip1 , or β-actin. The densitometry analyses of E2F-1, Cyclin D1, and p27 kip1 signals were shown underneath, and the arbitrary numbers indicate the intensities of each sample relative to controls, defined as 1.0. B C , The other half of the cells were collected for analysis of cell cycle distributions by flow cytometry as described in the Materials and Methods. Data show a representative of three independent experiments.

    Article Snippet: Immunohistochemistry Five micron thick paraffin sections were deparaffinized, antigens unmasked and immunohistochemically stained for Ki67 (Thermo Fisher Scientific; rabbit monoclonal SP6; cat# RM-9106-SO; dilution 1:500 in TBST + 1% BSA w/v), cleaved Caspase-3 (Cell Signaling Technology; rabbit polyclonal; cat#: 9661, 1:1000 in TBST + 1% BSA w/v), erbB2 (EMD Chemicals; mouse monoclonal 96G; cat#OP14T; dilution 1:500 in TBST + 1% BSA w/v), and erbB3 (Spring Bioscience, Pleasanton, CA; rabbit monoclonal SP71; cat# M3710; dilution 1:200 in TBST + 1% BSA w/v).

    Techniques: Western Blot, Flow Cytometry, Cytometry

    MM-121 enhances trastuzumab-mediated inactivation of Akt and growth inhibition in two erbB2+ breast cancer cell lines. A , SKBR3 and BT474 breast cancer cells were untreated or treated with either trastuzumab or MM-121 alone, or their combinations for 24 hrs. Cells were collected and subjected to western blot analyses of P-erbB2, erbB2, P-erbB3, erbB3, P-Akt, Akt, P-MAPK, MAPK, or β-actin. B , SKBR3 and BT474 cells were plated onto 96-well plates and incubated at 37°C with 5% CO2. After 24 hrs, the culture medium was replaced with 0.1 ml fresh medium containing 0.5% FBS or the same medium containing the indicated concentrations of trastuzumab in the absence (Trast) or presence (Trast + MM-121) of MM-121 (10 μg/ml) for another 72 hrs. The percentages of surviving cells from each cell line relative to controls, defined as 100% survival, were determined by reduction of MTS. Bars , SD. Data show a representative of three independent experiments.

    Journal: Molecular Cancer

    Article Title: The anti-erbB3 antibody MM-121/SAR256212 in combination with trastuzumab exerts potent antitumor activity against trastuzumab-resistant breast cancer cells

    doi: 10.1186/1476-4598-12-134

    Figure Lengend Snippet: MM-121 enhances trastuzumab-mediated inactivation of Akt and growth inhibition in two erbB2+ breast cancer cell lines. A , SKBR3 and BT474 breast cancer cells were untreated or treated with either trastuzumab or MM-121 alone, or their combinations for 24 hrs. Cells were collected and subjected to western blot analyses of P-erbB2, erbB2, P-erbB3, erbB3, P-Akt, Akt, P-MAPK, MAPK, or β-actin. B , SKBR3 and BT474 cells were plated onto 96-well plates and incubated at 37°C with 5% CO2. After 24 hrs, the culture medium was replaced with 0.1 ml fresh medium containing 0.5% FBS or the same medium containing the indicated concentrations of trastuzumab in the absence (Trast) or presence (Trast + MM-121) of MM-121 (10 μg/ml) for another 72 hrs. The percentages of surviving cells from each cell line relative to controls, defined as 100% survival, were determined by reduction of MTS. Bars , SD. Data show a representative of three independent experiments.

    Article Snippet: Immunohistochemistry Five micron thick paraffin sections were deparaffinized, antigens unmasked and immunohistochemically stained for Ki67 (Thermo Fisher Scientific; rabbit monoclonal SP6; cat# RM-9106-SO; dilution 1:500 in TBST + 1% BSA w/v), cleaved Caspase-3 (Cell Signaling Technology; rabbit polyclonal; cat#: 9661, 1:1000 in TBST + 1% BSA w/v), erbB2 (EMD Chemicals; mouse monoclonal 96G; cat#OP14T; dilution 1:500 in TBST + 1% BSA w/v), and erbB3 (Spring Bioscience, Pleasanton, CA; rabbit monoclonal SP71; cat# M3710; dilution 1:200 in TBST + 1% BSA w/v).

    Techniques: Inhibition, Western Blot, Incubation

    Translocation of ErbB2 into mitochondria is kinase dependent. (A) The mitochondrial fractions and whole cell lysates were isolated and Western blotting was performed on MDA-MB-435ErbB2 (435eb), MDA-MB-435ErbB2V695E (435VE) and MDA-MB-435ErbB2K753M (435KM) cells. MtHSP70 and α-Tubulin were loading controls. (B) Mitochondrial fractions and whole cell lysates were isolated and Western Blotting was performed to detect mtErbB2 in mitochondria following the treatment of MCF7ErbB2 cells with ErbB2 inhibitor AG825 at 10 uM or control (DMSO) for 24 h; and with Heregulin β1 (HRG) at 10 ng/ml or control (PBS) for 24 h (upper). Whole cell lysates were subjected to Western Blotting to examine the phosphorylation status of ErbB2 and the total ErbB2. α-Tubulin was loading control (lower). (C) MCF7 cells were treated with and without HRG at 10 ng/ml for 24 h. The mitochondrial fraction and the whole cell lysate were isolated for Western blotting. Prohibitin and α-Tubulin were loading controls. (D) Left, mitochondrial proteins were isolated from SKBR3 cells and immunoprecipitated with anti-ErbB2 or anti-Cox II antibodies. The immunoprecipitates were analyzed by Western blotting with anti-ErbB2 and anti-Cox II antibodies. IgG was negative control. Right, mitochondrial proteins were isolated from 231V, 231ErbB2WT, 231ErbB2Mito and 231ErbB2MitoKM cells and immunoprecipited with Cox II antibody or negative control IgG. Samples were loaded on a SDS–PAGE followed by Western Blotting analysis with Cox II antibody to show the Cox II protein level, and with P-Y20 antibody to show the tyrosine phosphorylation level on Cox II. (E) Cytochrome c oxidase activities were measured after the cells were treated with or without a cytochrome c oxidase inhibitor Potassium cyanide (KCN) in 231V, 231ErbB2WT, 231ErbB2Mito and 231ErbB2MitoKM cells. The relative activities of Cox II were calculated relative to the Cox II activity of 231V cells. (F) 231V, 231ErbB2WT, 231ErbB2Mito and 231ErbB2MitoKM cells were treated with or without Taxol at 80 nM for 24 h. Cytosolic, mitochondrial fractions and whole cell lysates were analyzed by Western blotting with cytochrome c antibodies. α-Tubulin and mtHSP70 were cytosol and mitochondrial markers and loading controls. Columns , mean of three independent experiments; bars , SE.*, P

    Journal: Nature communications

    Article Title: Receptor Tyrosine Kinase ErbB2 Translocates into Mitochondria and Regulates Cellular Metabolism

    doi: 10.1038/ncomms2236

    Figure Lengend Snippet: Translocation of ErbB2 into mitochondria is kinase dependent. (A) The mitochondrial fractions and whole cell lysates were isolated and Western blotting was performed on MDA-MB-435ErbB2 (435eb), MDA-MB-435ErbB2V695E (435VE) and MDA-MB-435ErbB2K753M (435KM) cells. MtHSP70 and α-Tubulin were loading controls. (B) Mitochondrial fractions and whole cell lysates were isolated and Western Blotting was performed to detect mtErbB2 in mitochondria following the treatment of MCF7ErbB2 cells with ErbB2 inhibitor AG825 at 10 uM or control (DMSO) for 24 h; and with Heregulin β1 (HRG) at 10 ng/ml or control (PBS) for 24 h (upper). Whole cell lysates were subjected to Western Blotting to examine the phosphorylation status of ErbB2 and the total ErbB2. α-Tubulin was loading control (lower). (C) MCF7 cells were treated with and without HRG at 10 ng/ml for 24 h. The mitochondrial fraction and the whole cell lysate were isolated for Western blotting. Prohibitin and α-Tubulin were loading controls. (D) Left, mitochondrial proteins were isolated from SKBR3 cells and immunoprecipitated with anti-ErbB2 or anti-Cox II antibodies. The immunoprecipitates were analyzed by Western blotting with anti-ErbB2 and anti-Cox II antibodies. IgG was negative control. Right, mitochondrial proteins were isolated from 231V, 231ErbB2WT, 231ErbB2Mito and 231ErbB2MitoKM cells and immunoprecipited with Cox II antibody or negative control IgG. Samples were loaded on a SDS–PAGE followed by Western Blotting analysis with Cox II antibody to show the Cox II protein level, and with P-Y20 antibody to show the tyrosine phosphorylation level on Cox II. (E) Cytochrome c oxidase activities were measured after the cells were treated with or without a cytochrome c oxidase inhibitor Potassium cyanide (KCN) in 231V, 231ErbB2WT, 231ErbB2Mito and 231ErbB2MitoKM cells. The relative activities of Cox II were calculated relative to the Cox II activity of 231V cells. (F) 231V, 231ErbB2WT, 231ErbB2Mito and 231ErbB2MitoKM cells were treated with or without Taxol at 80 nM for 24 h. Cytosolic, mitochondrial fractions and whole cell lysates were analyzed by Western blotting with cytochrome c antibodies. α-Tubulin and mtHSP70 were cytosol and mitochondrial markers and loading controls. Columns , mean of three independent experiments; bars , SE.*, P

    Article Snippet: Antibodies and Reagents The antibodies were purchased from: ErbB2 (OP15, Calbiochem), mtHSP70 (MA3-028, Thermo Scientific), VDAC1 (sc-58649, Santa Cruz), Bcl-2 (Rabbit mAb #2870, Cell Signaling), Integrin β (610467, BD Bioscience), Prohibitin (EP2803Y, Abcam), KDEL (sc-33806, Santa Cruz), EEA1(sc-130017, Santa Cruz), Golgi Complex (NB37-10UG, Calbiochem), LAMP2 (sc-18822, Santa Cruz), Lamin B1 (MA1-06103, Thermo Scientific), Mitochondrial Complex I subunit NDUFB8, Complex II subunit 30kDa, Complex III subunit Core 2, Complex IV subunit II, and ATP synthase subunit alpha antibodies mix were MitoProfile® Total OXPHOS Human WB Antibody Cocktail (MS601, Mitoscience). siRNAs for mtHSP70(1) (SASI_Hs01_00216924) and mtHSP70(2) (MISSION® siRNA SASI_Hs01_00216923) were purchased from Sigma.

    Techniques: Translocation Assay, Isolation, Western Blot, Multiple Displacement Amplification, Immunoprecipitation, Negative Control, SDS Page, Activity Assay

    Localization of ErbB2 in mitochondria. (A) Cytosolic, nuclear, mitochondrial, and plasma membrane proteins were isolated and subjected to SDS-PAGE followed by probing with indicated antibodies. Two exogenous ErbB2 overexpressing breast cancer cell lines MCF7/ErbB2 and MDA-MB-231 (left), and two natural ErbB2-positive breast cancer cell lines SKBR3 and BT474 (right) were used for Western Blotting. VDAC1 and prohibitin were mitochondrial markers; Integrin β1 and IGF1Rα were plasma membrane markers; α-Tubulin and ERK were cytoplasmic markers; KDEL was an ER marker; EEA1 was an early endosomes marker; Golgi complex was a marker for the detection of Golgi; LAMP2 was an lysosome marker and Lamin B1 was a nucleus marker. (B) MCF7 cells, mouse heart and liver tissues, and ErbB2 positive and negative breast cancer patient samples were analyzed by Western blotting. (C) Co-localization of ErbB2 and mitochondria. Mitochondria were stained with Mitotracker-Green in ErbB2 transfected MCF7 cells. The cells were fixed and incubated with antibodies against ErbB2 (mouse), followed by incubation of monoclonal mouse Anti–Cy3 antibody (red). Images were analyzed with Nikon NIS-Elements AR software. Green: mitochondria; Red: ErbB2; Yellow: Co-localization of ErbB2 and mitochondria. The lower panel contains images with a higher magnification. Scale bars: 20 μm. (D) Localization of ErbB2 inside mitochondria. Intact mitochondria of SKBR3 cells were isolated and treated with Protease K at 0.5 mg/ml for 15 min at room temperature. 1% Triton was added into the reaction and 2 mM PMSF was added to stop the reaction. Each reaction was started with an equal amount/volume of mitochondria lysate. Mitochondrial lysate then was loaded onto 8% SDS PAGE followed by blotting with anti-ErbB2, anti-BCL-2 and anti-Prohibitin antibodies.

    Journal: Nature communications

    Article Title: Receptor Tyrosine Kinase ErbB2 Translocates into Mitochondria and Regulates Cellular Metabolism

    doi: 10.1038/ncomms2236

    Figure Lengend Snippet: Localization of ErbB2 in mitochondria. (A) Cytosolic, nuclear, mitochondrial, and plasma membrane proteins were isolated and subjected to SDS-PAGE followed by probing with indicated antibodies. Two exogenous ErbB2 overexpressing breast cancer cell lines MCF7/ErbB2 and MDA-MB-231 (left), and two natural ErbB2-positive breast cancer cell lines SKBR3 and BT474 (right) were used for Western Blotting. VDAC1 and prohibitin were mitochondrial markers; Integrin β1 and IGF1Rα were plasma membrane markers; α-Tubulin and ERK were cytoplasmic markers; KDEL was an ER marker; EEA1 was an early endosomes marker; Golgi complex was a marker for the detection of Golgi; LAMP2 was an lysosome marker and Lamin B1 was a nucleus marker. (B) MCF7 cells, mouse heart and liver tissues, and ErbB2 positive and negative breast cancer patient samples were analyzed by Western blotting. (C) Co-localization of ErbB2 and mitochondria. Mitochondria were stained with Mitotracker-Green in ErbB2 transfected MCF7 cells. The cells were fixed and incubated with antibodies against ErbB2 (mouse), followed by incubation of monoclonal mouse Anti–Cy3 antibody (red). Images were analyzed with Nikon NIS-Elements AR software. Green: mitochondria; Red: ErbB2; Yellow: Co-localization of ErbB2 and mitochondria. The lower panel contains images with a higher magnification. Scale bars: 20 μm. (D) Localization of ErbB2 inside mitochondria. Intact mitochondria of SKBR3 cells were isolated and treated with Protease K at 0.5 mg/ml for 15 min at room temperature. 1% Triton was added into the reaction and 2 mM PMSF was added to stop the reaction. Each reaction was started with an equal amount/volume of mitochondria lysate. Mitochondrial lysate then was loaded onto 8% SDS PAGE followed by blotting with anti-ErbB2, anti-BCL-2 and anti-Prohibitin antibodies.

    Article Snippet: Antibodies and Reagents The antibodies were purchased from: ErbB2 (OP15, Calbiochem), mtHSP70 (MA3-028, Thermo Scientific), VDAC1 (sc-58649, Santa Cruz), Bcl-2 (Rabbit mAb #2870, Cell Signaling), Integrin β (610467, BD Bioscience), Prohibitin (EP2803Y, Abcam), KDEL (sc-33806, Santa Cruz), EEA1(sc-130017, Santa Cruz), Golgi Complex (NB37-10UG, Calbiochem), LAMP2 (sc-18822, Santa Cruz), Lamin B1 (MA1-06103, Thermo Scientific), Mitochondrial Complex I subunit NDUFB8, Complex II subunit 30kDa, Complex III subunit Core 2, Complex IV subunit II, and ATP synthase subunit alpha antibodies mix were MitoProfile® Total OXPHOS Human WB Antibody Cocktail (MS601, Mitoscience). siRNAs for mtHSP70(1) (SASI_Hs01_00216924) and mtHSP70(2) (MISSION® siRNA SASI_Hs01_00216923) were purchased from Sigma.

    Techniques: Isolation, SDS Page, Multiple Displacement Amplification, Western Blot, Marker, Staining, Transfection, Incubation, Software

    Analysis of the mechanisms of mtErbB2 mitochondrial localization. (A) MCF7 breast cancer cells were transfected with plasmids encoding either GFP alone, or GFP fused ErbB2 fragments GFP-646–689, GFP-623–645 or GFP-623–689. Cells were cultured and the florescent imaging was done as described under the “Methods.” Scale bars: 20 μm. (B) MDA-MB-231 cells were transfected with wild-type ErbB2 and ErbB2ΔMTS vectors and mitochondrial proteins were extracted. ErbB2 expression was measured by Western blotting analysis; α-Tubulin and mtHSP70 were loading controls. (C) Mitochondrial proteins were isolated from MDA-MB-231ErbB2 cells and immunoprecipitated with ErbB2 antibody. The immunoprecipitates were probed with anti-mtHSP70 and anti-ErbB2 (top left). Mitochondrial proteins from the same cells were precipitated with mtHSP70 antibody and the immunoprecipitates were probed with anti-mtHSP70 and anti-ErbB2 (top right). IgG was used as a negative control. Isolated mitochondrial proteins (input) were loaded as a positive control. Similar results were obtained using another breast cancer cell line, SKBR3 (bottom). (D) siRNA specific to mtHSP70 was transfected into MDA-MB-231ErbB2 cells. The cytoplasmic fraction (Cyto), mitochondrial fraction (Mito) and whole cell lysate (WCL) were separated for western blotting analysis (left). Cytochrome c oxidase subunit II and α-Tubulin were makers and loading controls for the mitochondrial fraction and the cytoplasmic fraction, respectively. The relative protein amounts of ErbB2 and mtHSP70 were calculated by determining the intensity of the protein bands followed by the normalization with loading controls (right). Experiments were repeated three times. Columns, mean of three independent experiments; bars, SE.

    Journal: Nature communications

    Article Title: Receptor Tyrosine Kinase ErbB2 Translocates into Mitochondria and Regulates Cellular Metabolism

    doi: 10.1038/ncomms2236

    Figure Lengend Snippet: Analysis of the mechanisms of mtErbB2 mitochondrial localization. (A) MCF7 breast cancer cells were transfected with plasmids encoding either GFP alone, or GFP fused ErbB2 fragments GFP-646–689, GFP-623–645 or GFP-623–689. Cells were cultured and the florescent imaging was done as described under the “Methods.” Scale bars: 20 μm. (B) MDA-MB-231 cells were transfected with wild-type ErbB2 and ErbB2ΔMTS vectors and mitochondrial proteins were extracted. ErbB2 expression was measured by Western blotting analysis; α-Tubulin and mtHSP70 were loading controls. (C) Mitochondrial proteins were isolated from MDA-MB-231ErbB2 cells and immunoprecipitated with ErbB2 antibody. The immunoprecipitates were probed with anti-mtHSP70 and anti-ErbB2 (top left). Mitochondrial proteins from the same cells were precipitated with mtHSP70 antibody and the immunoprecipitates were probed with anti-mtHSP70 and anti-ErbB2 (top right). IgG was used as a negative control. Isolated mitochondrial proteins (input) were loaded as a positive control. Similar results were obtained using another breast cancer cell line, SKBR3 (bottom). (D) siRNA specific to mtHSP70 was transfected into MDA-MB-231ErbB2 cells. The cytoplasmic fraction (Cyto), mitochondrial fraction (Mito) and whole cell lysate (WCL) were separated for western blotting analysis (left). Cytochrome c oxidase subunit II and α-Tubulin were makers and loading controls for the mitochondrial fraction and the cytoplasmic fraction, respectively. The relative protein amounts of ErbB2 and mtHSP70 were calculated by determining the intensity of the protein bands followed by the normalization with loading controls (right). Experiments were repeated three times. Columns, mean of three independent experiments; bars, SE.

    Article Snippet: Antibodies and Reagents The antibodies were purchased from: ErbB2 (OP15, Calbiochem), mtHSP70 (MA3-028, Thermo Scientific), VDAC1 (sc-58649, Santa Cruz), Bcl-2 (Rabbit mAb #2870, Cell Signaling), Integrin β (610467, BD Bioscience), Prohibitin (EP2803Y, Abcam), KDEL (sc-33806, Santa Cruz), EEA1(sc-130017, Santa Cruz), Golgi Complex (NB37-10UG, Calbiochem), LAMP2 (sc-18822, Santa Cruz), Lamin B1 (MA1-06103, Thermo Scientific), Mitochondrial Complex I subunit NDUFB8, Complex II subunit 30kDa, Complex III subunit Core 2, Complex IV subunit II, and ATP synthase subunit alpha antibodies mix were MitoProfile® Total OXPHOS Human WB Antibody Cocktail (MS601, Mitoscience). siRNAs for mtHSP70(1) (SASI_Hs01_00216924) and mtHSP70(2) (MISSION® siRNA SASI_Hs01_00216923) were purchased from Sigma.

    Techniques: Transfection, Cell Culture, Imaging, Multiple Displacement Amplification, Expressing, Western Blot, Isolation, Immunoprecipitation, Negative Control, Positive Control

    Translocation of ErbB2 into mitochondria contributes to trastuzumab resistance. (A) mtErbB2 is elevated in trastuzumab-resistant cancer cells. Mitochondrial proteins and whole cell lysates of BT474 and BT474 trastuzumab-resistant cells (BT474 HCP R) were isolated and analyzed by Western blotting. ATP Synthase and α-Tubulin were used as loading controls. (B) BT474 cells were treated with trastuzumab (HCP) at 10 ug/ml for 24 h and 48 h followed by the separation of cytosolic and mitochondrial fractions. Proteins from the cytoplasm, mitochondria and whole cell lysates were loaded onto gels and analyzed by Western blotting. mtHSP70 and α-Tubulin were used as loading controls (left). The relative protein amount of ErbB2 in the cellular fractions was calculated by detecting the intensity of the protein bands followed by normalization with loading controls (right). The experiments were repeated for three times. (C) mtErbB2-overexpressing cells are more resistant to trastuzumab. 231ErbB2WT, 231ErbB2Mito, 231ErbB2ΔMTS, 231ErbB2MitoKM and 231ErbB2WT cells transfected by siRNA to mtHSP70 were treated with trastuzumab at 100 ug/ml for 48 h and 72 h. The cell growth inhibition ratios were detected by a CellTiter 96 Aqueous One Solution Cell Proliferation Assay Kit. Columns, mean of three independent experiments; bars, SE. *, P

    Journal: Nature communications

    Article Title: Receptor Tyrosine Kinase ErbB2 Translocates into Mitochondria and Regulates Cellular Metabolism

    doi: 10.1038/ncomms2236

    Figure Lengend Snippet: Translocation of ErbB2 into mitochondria contributes to trastuzumab resistance. (A) mtErbB2 is elevated in trastuzumab-resistant cancer cells. Mitochondrial proteins and whole cell lysates of BT474 and BT474 trastuzumab-resistant cells (BT474 HCP R) were isolated and analyzed by Western blotting. ATP Synthase and α-Tubulin were used as loading controls. (B) BT474 cells were treated with trastuzumab (HCP) at 10 ug/ml for 24 h and 48 h followed by the separation of cytosolic and mitochondrial fractions. Proteins from the cytoplasm, mitochondria and whole cell lysates were loaded onto gels and analyzed by Western blotting. mtHSP70 and α-Tubulin were used as loading controls (left). The relative protein amount of ErbB2 in the cellular fractions was calculated by detecting the intensity of the protein bands followed by normalization with loading controls (right). The experiments were repeated for three times. (C) mtErbB2-overexpressing cells are more resistant to trastuzumab. 231ErbB2WT, 231ErbB2Mito, 231ErbB2ΔMTS, 231ErbB2MitoKM and 231ErbB2WT cells transfected by siRNA to mtHSP70 were treated with trastuzumab at 100 ug/ml for 48 h and 72 h. The cell growth inhibition ratios were detected by a CellTiter 96 Aqueous One Solution Cell Proliferation Assay Kit. Columns, mean of three independent experiments; bars, SE. *, P

    Article Snippet: Antibodies and Reagents The antibodies were purchased from: ErbB2 (OP15, Calbiochem), mtHSP70 (MA3-028, Thermo Scientific), VDAC1 (sc-58649, Santa Cruz), Bcl-2 (Rabbit mAb #2870, Cell Signaling), Integrin β (610467, BD Bioscience), Prohibitin (EP2803Y, Abcam), KDEL (sc-33806, Santa Cruz), EEA1(sc-130017, Santa Cruz), Golgi Complex (NB37-10UG, Calbiochem), LAMP2 (sc-18822, Santa Cruz), Lamin B1 (MA1-06103, Thermo Scientific), Mitochondrial Complex I subunit NDUFB8, Complex II subunit 30kDa, Complex III subunit Core 2, Complex IV subunit II, and ATP synthase subunit alpha antibodies mix were MitoProfile® Total OXPHOS Human WB Antibody Cocktail (MS601, Mitoscience). siRNAs for mtHSP70(1) (SASI_Hs01_00216924) and mtHSP70(2) (MISSION® siRNA SASI_Hs01_00216923) were purchased from Sigma.

    Techniques: Translocation Assay, Isolation, Western Blot, Transfection, Inhibition, Proliferation Assay

    Immunostaining of erbB2. SKBR3 and BT474 cells were treated with 50 µM TA for 24 hr and cells were fixed, immunostained with erbB2 or EEA1 antibodies and analyzed by confocal microscope and softwares as described in the Materials and Methods.

    Journal: Molecular cancer therapeutics

    Article Title: THE NSAID TOLFENAMIC ACID INHIBITS BT474 AND SKBR3 BREAST CANCER CELL AND TUMOR GROWTH BY REPRESSING erbB2 EXPRESSION

    doi: 10.1158/1535-7163.MCT-08-1097

    Figure Lengend Snippet: Immunostaining of erbB2. SKBR3 and BT474 cells were treated with 50 µM TA for 24 hr and cells were fixed, immunostained with erbB2 or EEA1 antibodies and analyzed by confocal microscope and softwares as described in the Materials and Methods.

    Article Snippet: Cells were incubated with anti-erbB2 antibody (1:80) or anti-EEA1 antibody (1:200) overnight and incubated with FITC-conjugated or Cy5-conjugated secondary antibody (1:200; Chemicon, Temecula, CA) for 1 hr.

    Techniques: Immunostaining, Microscopy

    Effects of TA on Sp, erbB2 and erbB2-dependent proteins. Sp protein expression in BT474 (A) and SKBR3 (B) cells treated with TA. Cells were treated with different concentrations of TA for up to 72 hr and whole cell lysates were analyzed by western blots

    Journal: Molecular cancer therapeutics

    Article Title: THE NSAID TOLFENAMIC ACID INHIBITS BT474 AND SKBR3 BREAST CANCER CELL AND TUMOR GROWTH BY REPRESSING erbB2 EXPRESSION

    doi: 10.1158/1535-7163.MCT-08-1097

    Figure Lengend Snippet: Effects of TA on Sp, erbB2 and erbB2-dependent proteins. Sp protein expression in BT474 (A) and SKBR3 (B) cells treated with TA. Cells were treated with different concentrations of TA for up to 72 hr and whole cell lysates were analyzed by western blots

    Article Snippet: Cells were incubated with anti-erbB2 antibody (1:80) or anti-EEA1 antibody (1:200) overnight and incubated with FITC-conjugated or Cy5-conjugated secondary antibody (1:200; Chemicon, Temecula, CA) for 1 hr.

    Techniques: Expressing, Western Blot

    TA decreases erbB2-dependent genes and erbB2 gene expression. Effects of TA on cyclin D1/p27 (A) and erbB2 (B) protein levels. BT474 and SKBR3 cells were treated with TA alone or TA in combination with 2 µM lactacystin for 48 or 72 hr as indicated,

    Journal: Molecular cancer therapeutics

    Article Title: THE NSAID TOLFENAMIC ACID INHIBITS BT474 AND SKBR3 BREAST CANCER CELL AND TUMOR GROWTH BY REPRESSING erbB2 EXPRESSION

    doi: 10.1158/1535-7163.MCT-08-1097

    Figure Lengend Snippet: TA decreases erbB2-dependent genes and erbB2 gene expression. Effects of TA on cyclin D1/p27 (A) and erbB2 (B) protein levels. BT474 and SKBR3 cells were treated with TA alone or TA in combination with 2 µM lactacystin for 48 or 72 hr as indicated,

    Article Snippet: Cells were incubated with anti-erbB2 antibody (1:80) or anti-EEA1 antibody (1:200) overnight and incubated with FITC-conjugated or Cy5-conjugated secondary antibody (1:200; Chemicon, Temecula, CA) for 1 hr.

    Techniques: Expressing

    Effects of TA on erbB2 expression and transcriptional regulatory proteins. Effects of cycloheximide (A) and actinomycin D (B) on erbB2 mRNA levels and stability in cells treated with TA. BT474 and SKBR3 cells were treated with 50 µM TA alone or

    Journal: Molecular cancer therapeutics

    Article Title: THE NSAID TOLFENAMIC ACID INHIBITS BT474 AND SKBR3 BREAST CANCER CELL AND TUMOR GROWTH BY REPRESSING erbB2 EXPRESSION

    doi: 10.1158/1535-7163.MCT-08-1097

    Figure Lengend Snippet: Effects of TA on erbB2 expression and transcriptional regulatory proteins. Effects of cycloheximide (A) and actinomycin D (B) on erbB2 mRNA levels and stability in cells treated with TA. BT474 and SKBR3 cells were treated with 50 µM TA alone or

    Article Snippet: Cells were incubated with anti-erbB2 antibody (1:80) or anti-EEA1 antibody (1:200) overnight and incubated with FITC-conjugated or Cy5-conjugated secondary antibody (1:200; Chemicon, Temecula, CA) for 1 hr.

    Techniques: Expressing

    Expression of miR‐205 in MCF 10A‐Raf CAAX cells treated with ERK inhibitor. (A) Western blot analysis of MCF 10A‐Raf CAAX , MCF 10A‐ErbB2, and MCF 10A‐neo cells with anti‐Raf‐1 antibodies. β‐Actin was used as a control for loading. (B) Real‐time quantitative RT ‐ PCR analysis of miR‐205 expression in MCF 10A‐Raf CAAX and MCF 10A‐neo cells. Data are mean ± SEM of three independent experiments. (C) Real‐time quantitative RT ‐ PCR analysis of miR‐205 expression in MCF 10A‐neo and MCF 10A‐Raf CAAX cells treated with MEK inhibitor U0126 (10 μ m ) or PD 98059 (20 μ m ), Raf1 kinase inhibitor ZM ‐336372 (1 μ m ), or ERK inhibitor SCH 772984 (1 μ m ) for 48 h. Data are mean ± SEM of three independent experiments. * P

    Journal: FEBS Open Bio

    Article Title: ErbB2 signaling epigenetically suppresses micro RNA‐205 transcription via Ras/Raf/ MEK/ ERK pathway in breast cancer

    doi: 10.1002/2211-5463.12256

    Figure Lengend Snippet: Expression of miR‐205 in MCF 10A‐Raf CAAX cells treated with ERK inhibitor. (A) Western blot analysis of MCF 10A‐Raf CAAX , MCF 10A‐ErbB2, and MCF 10A‐neo cells with anti‐Raf‐1 antibodies. β‐Actin was used as a control for loading. (B) Real‐time quantitative RT ‐ PCR analysis of miR‐205 expression in MCF 10A‐Raf CAAX and MCF 10A‐neo cells. Data are mean ± SEM of three independent experiments. (C) Real‐time quantitative RT ‐ PCR analysis of miR‐205 expression in MCF 10A‐neo and MCF 10A‐Raf CAAX cells treated with MEK inhibitor U0126 (10 μ m ) or PD 98059 (20 μ m ), Raf1 kinase inhibitor ZM ‐336372 (1 μ m ), or ERK inhibitor SCH 772984 (1 μ m ) for 48 h. Data are mean ± SEM of three independent experiments. * P

    Article Snippet: The following antibodies were used: 53, a mouse monoclonal antibody to Raf‐1 (BD Biosciences, San Diego, CA, USA); H‐300, a rabbit polyclonal antibody to DNMT1 (Santa Cruz Biotechnology, Dallas, TX, USA); H‐295, a rabbit polyclonal antibody to DNMT3a (Santa Cruz Biotechnology); H‐230, a rabbit polyclonal antibody to DNMT3b (Santa Cruz Biotechnology); c‐ErbB2 (Oncogene Research Products, San Diego, CA, USA); ab4767, a rabbit polyclonal antibody to Raf1 (phospho‐S621) (Abcam, Cambridge, UK); L38C12, a mouse monoclonal antibody to MEK1/2 (Cell Signaling Technology, Danvers, MA, USA); 41G9, a rabbit monoclonal antibody to phospho‐MEK1/2 (Cell Signaling Technology); L34F12, a mouse monoclonal antibody to p44/42 MAPK (Erk1/2) (Cell Signaling Technology); D13.14.4E, a rabbit monoclonal antibody to phospho‐p44/42 MAPK (Erk1/2) (Cell Signaling Technology); MAB1501, a mouse monoclonal antibody to actin (Millipore, Billerica, MA, USA), as a protein loading control; and horseradish peroxidase‐conjugated goat antibodies to mouse and rabbit IgG (Santa Cruz Biotechnology).

    Techniques: Expressing, Western Blot, Quantitative RT-PCR

    ErbB2 signaling via Ras/Raf/ MEK / ERK pathway induces hypermethylation of miR‐205 promoters. (A) Graphical depiction of the MIR 205 host gene ( MIR 205 HG ). Blue two‐way arrows indicate the promoter regions analyzed by bisulfite sequencing (A: MIR 205 HG , B: MIR 205 ). Red bent arrows indicate the putative transcription start sites ( TSS ) of MIR 205 HG and MIR 205 genes. Blue rectangles represent exons of the MIR 205 HG gene. Green rectangle represents the MIR 205 locus. (B, C) Bisulfite sequencing analysis of promoter A (B) and promoter B (C). White circles indicate nonmethylated cytosine and black circles indicate methylated cytosine at individual CpG sites. The graphs show the percentage of methylation at individual CpG sites. (D) Real‐time quantitative RT ‐ PCR analysis of MCF 10A‐Raf CAAX , MCF 10A‐ErbB2, and MCF 10A‐neo cells for MIR 205 HG . Data are mean ± SEM of three independent experiments. * P

    Journal: FEBS Open Bio

    Article Title: ErbB2 signaling epigenetically suppresses micro RNA‐205 transcription via Ras/Raf/ MEK/ ERK pathway in breast cancer

    doi: 10.1002/2211-5463.12256

    Figure Lengend Snippet: ErbB2 signaling via Ras/Raf/ MEK / ERK pathway induces hypermethylation of miR‐205 promoters. (A) Graphical depiction of the MIR 205 host gene ( MIR 205 HG ). Blue two‐way arrows indicate the promoter regions analyzed by bisulfite sequencing (A: MIR 205 HG , B: MIR 205 ). Red bent arrows indicate the putative transcription start sites ( TSS ) of MIR 205 HG and MIR 205 genes. Blue rectangles represent exons of the MIR 205 HG gene. Green rectangle represents the MIR 205 locus. (B, C) Bisulfite sequencing analysis of promoter A (B) and promoter B (C). White circles indicate nonmethylated cytosine and black circles indicate methylated cytosine at individual CpG sites. The graphs show the percentage of methylation at individual CpG sites. (D) Real‐time quantitative RT ‐ PCR analysis of MCF 10A‐Raf CAAX , MCF 10A‐ErbB2, and MCF 10A‐neo cells for MIR 205 HG . Data are mean ± SEM of three independent experiments. * P

    Article Snippet: The following antibodies were used: 53, a mouse monoclonal antibody to Raf‐1 (BD Biosciences, San Diego, CA, USA); H‐300, a rabbit polyclonal antibody to DNMT1 (Santa Cruz Biotechnology, Dallas, TX, USA); H‐295, a rabbit polyclonal antibody to DNMT3a (Santa Cruz Biotechnology); H‐230, a rabbit polyclonal antibody to DNMT3b (Santa Cruz Biotechnology); c‐ErbB2 (Oncogene Research Products, San Diego, CA, USA); ab4767, a rabbit polyclonal antibody to Raf1 (phospho‐S621) (Abcam, Cambridge, UK); L38C12, a mouse monoclonal antibody to MEK1/2 (Cell Signaling Technology, Danvers, MA, USA); 41G9, a rabbit monoclonal antibody to phospho‐MEK1/2 (Cell Signaling Technology); L34F12, a mouse monoclonal antibody to p44/42 MAPK (Erk1/2) (Cell Signaling Technology); D13.14.4E, a rabbit monoclonal antibody to phospho‐p44/42 MAPK (Erk1/2) (Cell Signaling Technology); MAB1501, a mouse monoclonal antibody to actin (Millipore, Billerica, MA, USA), as a protein loading control; and horseradish peroxidase‐conjugated goat antibodies to mouse and rabbit IgG (Santa Cruz Biotechnology).

    Techniques: Methylation Sequencing, Methylation, Quantitative RT-PCR

    Luciferase reporter analysis of the miR‐205 promoters. MCF 10A‐Raf CAAX , MCF 10A‐ErbB2, and MCF 10A‐neo cells were transiently cotransfected with pGL 4.74 Renilla luciferase plasmid and reporter plasmid of promoter A (A) or with pGL 4.74 Renilla luciferase plasmid and reporter plasmid of promoter B (B). At 36 h post‐transfection, luciferase activities were measured. Data are normalized to pGL 4.74 Renilla luciferase plasmid control and represented as mean ± SEM of three independent experiments. * P

    Journal: FEBS Open Bio

    Article Title: ErbB2 signaling epigenetically suppresses micro RNA‐205 transcription via Ras/Raf/ MEK/ ERK pathway in breast cancer

    doi: 10.1002/2211-5463.12256

    Figure Lengend Snippet: Luciferase reporter analysis of the miR‐205 promoters. MCF 10A‐Raf CAAX , MCF 10A‐ErbB2, and MCF 10A‐neo cells were transiently cotransfected with pGL 4.74 Renilla luciferase plasmid and reporter plasmid of promoter A (A) or with pGL 4.74 Renilla luciferase plasmid and reporter plasmid of promoter B (B). At 36 h post‐transfection, luciferase activities were measured. Data are normalized to pGL 4.74 Renilla luciferase plasmid control and represented as mean ± SEM of three independent experiments. * P

    Article Snippet: The following antibodies were used: 53, a mouse monoclonal antibody to Raf‐1 (BD Biosciences, San Diego, CA, USA); H‐300, a rabbit polyclonal antibody to DNMT1 (Santa Cruz Biotechnology, Dallas, TX, USA); H‐295, a rabbit polyclonal antibody to DNMT3a (Santa Cruz Biotechnology); H‐230, a rabbit polyclonal antibody to DNMT3b (Santa Cruz Biotechnology); c‐ErbB2 (Oncogene Research Products, San Diego, CA, USA); ab4767, a rabbit polyclonal antibody to Raf1 (phospho‐S621) (Abcam, Cambridge, UK); L38C12, a mouse monoclonal antibody to MEK1/2 (Cell Signaling Technology, Danvers, MA, USA); 41G9, a rabbit monoclonal antibody to phospho‐MEK1/2 (Cell Signaling Technology); L34F12, a mouse monoclonal antibody to p44/42 MAPK (Erk1/2) (Cell Signaling Technology); D13.14.4E, a rabbit monoclonal antibody to phospho‐p44/42 MAPK (Erk1/2) (Cell Signaling Technology); MAB1501, a mouse monoclonal antibody to actin (Millipore, Billerica, MA, USA), as a protein loading control; and horseradish peroxidase‐conjugated goat antibodies to mouse and rabbit IgG (Santa Cruz Biotechnology).

    Techniques: Luciferase, Plasmid Preparation, Transfection

    Effects of DNA demethylation on miR‐205 expression and expression of DNMT family in MCF 10A‐neo, MCF 10A‐ErbB2, and MCF 10A‐Raf CAAX cells. (A) Real‐time quantitative RT ‐ PCR analysis of miR‐205 in MCF 10A‐Raf CAAX , MCF 10A‐ErbB2, and MCF 10A‐neo cells treated with the DNA methylation inhibitor. Cells were treated with 5‐aza‐2′‐deoxycytidine (5 μ m ) for 48 h. Data are normalized to vehicle control and represented as mean ± SEM of three independent experiments. * P

    Journal: FEBS Open Bio

    Article Title: ErbB2 signaling epigenetically suppresses micro RNA‐205 transcription via Ras/Raf/ MEK/ ERK pathway in breast cancer

    doi: 10.1002/2211-5463.12256

    Figure Lengend Snippet: Effects of DNA demethylation on miR‐205 expression and expression of DNMT family in MCF 10A‐neo, MCF 10A‐ErbB2, and MCF 10A‐Raf CAAX cells. (A) Real‐time quantitative RT ‐ PCR analysis of miR‐205 in MCF 10A‐Raf CAAX , MCF 10A‐ErbB2, and MCF 10A‐neo cells treated with the DNA methylation inhibitor. Cells were treated with 5‐aza‐2′‐deoxycytidine (5 μ m ) for 48 h. Data are normalized to vehicle control and represented as mean ± SEM of three independent experiments. * P

    Article Snippet: The following antibodies were used: 53, a mouse monoclonal antibody to Raf‐1 (BD Biosciences, San Diego, CA, USA); H‐300, a rabbit polyclonal antibody to DNMT1 (Santa Cruz Biotechnology, Dallas, TX, USA); H‐295, a rabbit polyclonal antibody to DNMT3a (Santa Cruz Biotechnology); H‐230, a rabbit polyclonal antibody to DNMT3b (Santa Cruz Biotechnology); c‐ErbB2 (Oncogene Research Products, San Diego, CA, USA); ab4767, a rabbit polyclonal antibody to Raf1 (phospho‐S621) (Abcam, Cambridge, UK); L38C12, a mouse monoclonal antibody to MEK1/2 (Cell Signaling Technology, Danvers, MA, USA); 41G9, a rabbit monoclonal antibody to phospho‐MEK1/2 (Cell Signaling Technology); L34F12, a mouse monoclonal antibody to p44/42 MAPK (Erk1/2) (Cell Signaling Technology); D13.14.4E, a rabbit monoclonal antibody to phospho‐p44/42 MAPK (Erk1/2) (Cell Signaling Technology); MAB1501, a mouse monoclonal antibody to actin (Millipore, Billerica, MA, USA), as a protein loading control; and horseradish peroxidase‐conjugated goat antibodies to mouse and rabbit IgG (Santa Cruz Biotechnology).

    Techniques: Expressing, Quantitative RT-PCR, DNA Methylation Assay

    Expression of miR‐205 in MCF 10A‐ErbB2, SKBR 3, and MDA ‐ MB ‐453 cells treated with ErbB2 signaling pathway inhibitors. Real‐time quantitative RT ‐ PCR analysis of miR‐205 expression in MCF 10A‐ErbB2, SKBR 3, and MDA ‐ MB ‐453 cells treated with the indicated inhibitors. (A, D) Cells were treated with PI 3K inhibitor LY 294002 (50 μ m ), p38 MAPK inhibitor SB 203580 (10 μ m ), MEK inhibitor U0126 (10 μ m ), or PD 98059 (20 μ m ) for 48 h. (B, D) Cells were treated with Raf‐1 kinase inhibitor ZM ‐336372 (1–5 μ m ) or ERK inhibitor SCH 772984 (1 μ m ) for 48 h. Data are normalized to DMSO control and represented as mean ± SEM of three independent experiments. * P

    Journal: FEBS Open Bio

    Article Title: ErbB2 signaling epigenetically suppresses micro RNA‐205 transcription via Ras/Raf/ MEK/ ERK pathway in breast cancer

    doi: 10.1002/2211-5463.12256

    Figure Lengend Snippet: Expression of miR‐205 in MCF 10A‐ErbB2, SKBR 3, and MDA ‐ MB ‐453 cells treated with ErbB2 signaling pathway inhibitors. Real‐time quantitative RT ‐ PCR analysis of miR‐205 expression in MCF 10A‐ErbB2, SKBR 3, and MDA ‐ MB ‐453 cells treated with the indicated inhibitors. (A, D) Cells were treated with PI 3K inhibitor LY 294002 (50 μ m ), p38 MAPK inhibitor SB 203580 (10 μ m ), MEK inhibitor U0126 (10 μ m ), or PD 98059 (20 μ m ) for 48 h. (B, D) Cells were treated with Raf‐1 kinase inhibitor ZM ‐336372 (1–5 μ m ) or ERK inhibitor SCH 772984 (1 μ m ) for 48 h. Data are normalized to DMSO control and represented as mean ± SEM of three independent experiments. * P

    Article Snippet: The following antibodies were used: 53, a mouse monoclonal antibody to Raf‐1 (BD Biosciences, San Diego, CA, USA); H‐300, a rabbit polyclonal antibody to DNMT1 (Santa Cruz Biotechnology, Dallas, TX, USA); H‐295, a rabbit polyclonal antibody to DNMT3a (Santa Cruz Biotechnology); H‐230, a rabbit polyclonal antibody to DNMT3b (Santa Cruz Biotechnology); c‐ErbB2 (Oncogene Research Products, San Diego, CA, USA); ab4767, a rabbit polyclonal antibody to Raf1 (phospho‐S621) (Abcam, Cambridge, UK); L38C12, a mouse monoclonal antibody to MEK1/2 (Cell Signaling Technology, Danvers, MA, USA); 41G9, a rabbit monoclonal antibody to phospho‐MEK1/2 (Cell Signaling Technology); L34F12, a mouse monoclonal antibody to p44/42 MAPK (Erk1/2) (Cell Signaling Technology); D13.14.4E, a rabbit monoclonal antibody to phospho‐p44/42 MAPK (Erk1/2) (Cell Signaling Technology); MAB1501, a mouse monoclonal antibody to actin (Millipore, Billerica, MA, USA), as a protein loading control; and horseradish peroxidase‐conjugated goat antibodies to mouse and rabbit IgG (Santa Cruz Biotechnology).

    Techniques: Expressing, Multiple Displacement Amplification, Quantitative RT-PCR

    Schematic representation showing downregulation of miR‐205 by ErbB2 signaling via Ras/Raf/ MEK / ERK pathway. ErbB2 signaling via Ras/Raf/ MEK / ERK pathway leads to hypermethylation of the CpG‐rich regions of the promoters of MIR 205 HG and MIR 205 by inducing DNMT family, resulting in downregulation of miR‐205 expression.

    Journal: FEBS Open Bio

    Article Title: ErbB2 signaling epigenetically suppresses micro RNA‐205 transcription via Ras/Raf/ MEK/ ERK pathway in breast cancer

    doi: 10.1002/2211-5463.12256

    Figure Lengend Snippet: Schematic representation showing downregulation of miR‐205 by ErbB2 signaling via Ras/Raf/ MEK / ERK pathway. ErbB2 signaling via Ras/Raf/ MEK / ERK pathway leads to hypermethylation of the CpG‐rich regions of the promoters of MIR 205 HG and MIR 205 by inducing DNMT family, resulting in downregulation of miR‐205 expression.

    Article Snippet: The following antibodies were used: 53, a mouse monoclonal antibody to Raf‐1 (BD Biosciences, San Diego, CA, USA); H‐300, a rabbit polyclonal antibody to DNMT1 (Santa Cruz Biotechnology, Dallas, TX, USA); H‐295, a rabbit polyclonal antibody to DNMT3a (Santa Cruz Biotechnology); H‐230, a rabbit polyclonal antibody to DNMT3b (Santa Cruz Biotechnology); c‐ErbB2 (Oncogene Research Products, San Diego, CA, USA); ab4767, a rabbit polyclonal antibody to Raf1 (phospho‐S621) (Abcam, Cambridge, UK); L38C12, a mouse monoclonal antibody to MEK1/2 (Cell Signaling Technology, Danvers, MA, USA); 41G9, a rabbit monoclonal antibody to phospho‐MEK1/2 (Cell Signaling Technology); L34F12, a mouse monoclonal antibody to p44/42 MAPK (Erk1/2) (Cell Signaling Technology); D13.14.4E, a rabbit monoclonal antibody to phospho‐p44/42 MAPK (Erk1/2) (Cell Signaling Technology); MAB1501, a mouse monoclonal antibody to actin (Millipore, Billerica, MA, USA), as a protein loading control; and horseradish peroxidase‐conjugated goat antibodies to mouse and rabbit IgG (Santa Cruz Biotechnology).

    Techniques: Expressing

    Expression of neuregulin, ErbB2, and AKT in injured sciatic nerves from WT and αBC −/− mice. Western blot levels and ImageJ analysis of ( A ) neuregulin 1 Types I and III, ( B ) ErbB2, and ( C ) AKT in WT and αBC −/− animals before injury (N) and at various time points (3, 5, 7, 14, and 28 d) after crush damage (one experiment; n = 4 per group). Displayed are two animals per time point, with each quantification time point consisting of four animals. All data represent mean ± SEM. * P

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    Article Title: AlphaB-crystallin regulates remyelination after peripheral nerve injury

    doi: 10.1073/pnas.1612136114

    Figure Lengend Snippet: Expression of neuregulin, ErbB2, and AKT in injured sciatic nerves from WT and αBC −/− mice. Western blot levels and ImageJ analysis of ( A ) neuregulin 1 Types I and III, ( B ) ErbB2, and ( C ) AKT in WT and αBC −/− animals before injury (N) and at various time points (3, 5, 7, 14, and 28 d) after crush damage (one experiment; n = 4 per group). Displayed are two animals per time point, with each quantification time point consisting of four animals. All data represent mean ± SEM. * P

    Article Snippet: Membranes were immunoblotted overnight at 4 °C with the following primary antibodies: Ms anti–GAP-43 (MAB347; 1:400; Millipore), Rb anti-αBC (ABN185; 1:1,000; EMD Millipore), Rb anti-actin (A2006; 1:1,000; Sigma-Aldrich), Rb neuregulin 1 Type I (sc-348; 1:500; Santa Cruz), Ms neuregulin 1 Type III (MABN42; 1:1,000; Millipore), Sh ErbB2 (AF5176; 1:1,000; R & D), Ms p-ErbB2 (04–294; 1:1,000; Millipore), Rb AKT (9272; 1:1,000; Cell Signaling), Rb p-AKT (4060; 1:1,000; Cell Signaling), Rb p38 (9212; 1:1,000; Cell Signaling), Ms p-p38 (9216; 1:2,000; Cell Signaling), Rb ERK (9102; 1:1,000; Cell Signaling ), Rb p-ERK (9101; 1:1,000; Cell Signaling ), Rb JNK (9252; 1:1,000; Cell Signaling ), and Rb p-JNK (9251; 1:1,000; Cell Signaling).

    Techniques: Expressing, Mouse Assay, Western Blot

    The effects of CagA on cell growth and HER-2 expression. (A) Cell growth as measured by the MTT assay in AGS, MKN1, and HFE-145 cells after transfection. CagA transfected cells showed significantly increased growth. (B C) Effect of CagA on HER-2 DNA copy number and mRNA expression in AGS, MKN1, and HFE-145 cells. Ectopic CagA expression induced an increase of HER-2 DNA copy number and mRNA transcript expression. (D) HER-2 protein expression measured by Western blot in CagA transfected AGS, MKN1, and HFE-145 cells. Increased expression of the HER-2 protein was observed in CagA transfected cells compared to those in mock (vector + Lipofectamine) transfected cells. (E F) The gastric mucosae of H. pylori infected C57BL/6 mice showed increased HER-2 DNA copy number and protein expression. (G) CagA or H 2 O 2 treatment significantly increased ROS production, but treatment with the antioxidant TEMPOL reverted the CagA- and H 2 O 2 -induced ROS level in AGS,MKN1, and HFE-145 cells. (H) CagA or H 2 O 2 treatment significantly increased HER-2 DNA copy number, whereas TEMPOL reverted the H 2 O 2 -induced HER-2 copy number change in AGS cells.

    Journal: Gene

    Article Title: The effect of Helicobacter pylori CagA on the HER-2 copy number and expression in gastric cancer

    doi: 10.1016/j.gene.2014.05.064

    Figure Lengend Snippet: The effects of CagA on cell growth and HER-2 expression. (A) Cell growth as measured by the MTT assay in AGS, MKN1, and HFE-145 cells after transfection. CagA transfected cells showed significantly increased growth. (B C) Effect of CagA on HER-2 DNA copy number and mRNA expression in AGS, MKN1, and HFE-145 cells. Ectopic CagA expression induced an increase of HER-2 DNA copy number and mRNA transcript expression. (D) HER-2 protein expression measured by Western blot in CagA transfected AGS, MKN1, and HFE-145 cells. Increased expression of the HER-2 protein was observed in CagA transfected cells compared to those in mock (vector + Lipofectamine) transfected cells. (E F) The gastric mucosae of H. pylori infected C57BL/6 mice showed increased HER-2 DNA copy number and protein expression. (G) CagA or H 2 O 2 treatment significantly increased ROS production, but treatment with the antioxidant TEMPOL reverted the CagA- and H 2 O 2 -induced ROS level in AGS,MKN1, and HFE-145 cells. (H) CagA or H 2 O 2 treatment significantly increased HER-2 DNA copy number, whereas TEMPOL reverted the H 2 O 2 -induced HER-2 copy number change in AGS cells.

    Article Snippet: The sections were incubated overnight at 4 °C with HER-2 antibody (1/100; Sigma, St. Louis, MO, USA).

    Techniques: Expressing, MTT Assay, Transfection, Western Blot, Plasmid Preparation, Infection, Mouse Assay

    Association between the presence of CagA and HER-2 status in GC tissues. (A B) The presence of CagA was closely associated with the HER-2 DNA amplification and mRNA overexpression. (C D) CagA predicted HER-2 DNA amplification and mRNA overexpression with an area under the ROC curve (AUC) value of 0.8733 and 0.9457, respectively. Neg, negative; Pos, positive.

    Journal: Gene

    Article Title: The effect of Helicobacter pylori CagA on the HER-2 copy number and expression in gastric cancer

    doi: 10.1016/j.gene.2014.05.064

    Figure Lengend Snippet: Association between the presence of CagA and HER-2 status in GC tissues. (A B) The presence of CagA was closely associated with the HER-2 DNA amplification and mRNA overexpression. (C D) CagA predicted HER-2 DNA amplification and mRNA overexpression with an area under the ROC curve (AUC) value of 0.8733 and 0.9457, respectively. Neg, negative; Pos, positive.

    Article Snippet: The sections were incubated overnight at 4 °C with HER-2 antibody (1/100; Sigma, St. Louis, MO, USA).

    Techniques: Amplification, Over Expression

    Detection of erbB-2 and erbB-4 mRNAs and absence of erbB-3 mRNA in isolated hypothalamic ( Hypo ) and cerebrocortical ( CTX ) astrocytes (Astro.), as assessed by RNase protection assay. M , 32 P-UTP-labeled RNA molecular size marker; UP , undigested cRNA probes; DP , digested probes; cyclo , cyclophilin.

    Journal: The Journal of Neuroscience

    Article Title: Neuregulins Signaling via a Glial erbB-2–erbB-4 Receptor Complex Contribute to the Neuroendocrine Control of Mammalian Sexual Development

    doi: 10.1523/JNEUROSCI.19-22-09913.1999

    Figure Lengend Snippet: Detection of erbB-2 and erbB-4 mRNAs and absence of erbB-3 mRNA in isolated hypothalamic ( Hypo ) and cerebrocortical ( CTX ) astrocytes (Astro.), as assessed by RNase protection assay. M , 32 P-UTP-labeled RNA molecular size marker; UP , undigested cRNA probes; DP , digested probes; cyclo , cyclophilin.

    Article Snippet: Because the medial basal hypothalamic–median eminence region showed an abundance of erbB-2 and erbB-4 mRNA transcripts, we examined this region for the presence of erbB-2- and erbB-4-immunoreactive cells using immunohistofluorescence followed by confocal microscopy.

    Techniques: Isolation, Rnase Protection Assay, Labeling, Marker

    A , Left to right , Tyrosine phosphorylation of erbB-1 and erbB-2 receptors in MCF-7 cells induced by EGF and NDF-β2, and phosphorylation of erbB1, erbB-2, and erbB-4 receptors in hypothalamic astrocytes ( H.A. ) induced by TGFα or NDF-β2. The cells were exposed for 5 min to each ligand (100 ng/ml) before lysis. The erbB receptors were immunoprecipitated ( IP ) with antibodies specific to each protein, and the tyrosine phosphorylated receptors were identified by Western blots using phosphotyrosine antibodies. B , Left to right , Phosphorylation of erbB-1 and transphosphorylation of erbB-2 in cerebrocortical astrocytes ( C.A. ) by TGFα; inability of NDF-β 2 to phosphorylate erbB-2 in cortical astrocytes; phosphorylation of erbB-2 in hypothalamic astrocytes by NDF-β2; and effectiveness of NDF-β2 to induce erbB-2 phosphorylation in cortical astrocytes after transient overexpression ( Transf. ) of erbB-4.

    Journal: The Journal of Neuroscience

    Article Title: Neuregulins Signaling via a Glial erbB-2–erbB-4 Receptor Complex Contribute to the Neuroendocrine Control of Mammalian Sexual Development

    doi: 10.1523/JNEUROSCI.19-22-09913.1999

    Figure Lengend Snippet: A , Left to right , Tyrosine phosphorylation of erbB-1 and erbB-2 receptors in MCF-7 cells induced by EGF and NDF-β2, and phosphorylation of erbB1, erbB-2, and erbB-4 receptors in hypothalamic astrocytes ( H.A. ) induced by TGFα or NDF-β2. The cells were exposed for 5 min to each ligand (100 ng/ml) before lysis. The erbB receptors were immunoprecipitated ( IP ) with antibodies specific to each protein, and the tyrosine phosphorylated receptors were identified by Western blots using phosphotyrosine antibodies. B , Left to right , Phosphorylation of erbB-1 and transphosphorylation of erbB-2 in cerebrocortical astrocytes ( C.A. ) by TGFα; inability of NDF-β 2 to phosphorylate erbB-2 in cortical astrocytes; phosphorylation of erbB-2 in hypothalamic astrocytes by NDF-β2; and effectiveness of NDF-β2 to induce erbB-2 phosphorylation in cortical astrocytes after transient overexpression ( Transf. ) of erbB-4.

    Article Snippet: Because the medial basal hypothalamic–median eminence region showed an abundance of erbB-2 and erbB-4 mRNA transcripts, we examined this region for the presence of erbB-2- and erbB-4-immunoreactive cells using immunohistofluorescence followed by confocal microscopy.

    Techniques: Lysis, Immunoprecipitation, Western Blot, Over Expression

    Heterodimerization of erbB-4 with erbB-2 receptors in hypothalamic astrocytes. After exposure to NDF-β2 (500 ng/ml, 3 min), the cells were exposed to the cross-linker BS 3 , lysed, immunoprecipitated ( IP ) with either erbB-2 or erbB-4 antibodies, and blotted ( IB ) with erbB-2 or erbB-4 antibodies. Notice the formation of a high molecular weight complex containing both erbB-2 ( left and middle ) and erbB-4 ( right ).

    Journal: The Journal of Neuroscience

    Article Title: Neuregulins Signaling via a Glial erbB-2–erbB-4 Receptor Complex Contribute to the Neuroendocrine Control of Mammalian Sexual Development

    doi: 10.1523/JNEUROSCI.19-22-09913.1999

    Figure Lengend Snippet: Heterodimerization of erbB-4 with erbB-2 receptors in hypothalamic astrocytes. After exposure to NDF-β2 (500 ng/ml, 3 min), the cells were exposed to the cross-linker BS 3 , lysed, immunoprecipitated ( IP ) with either erbB-2 or erbB-4 antibodies, and blotted ( IB ) with erbB-2 or erbB-4 antibodies. Notice the formation of a high molecular weight complex containing both erbB-2 ( left and middle ) and erbB-4 ( right ).

    Article Snippet: Because the medial basal hypothalamic–median eminence region showed an abundance of erbB-2 and erbB-4 mRNA transcripts, we examined this region for the presence of erbB-2- and erbB-4-immunoreactive cells using immunohistofluorescence followed by confocal microscopy.

    Techniques: Immunoprecipitation, Molecular Weight

    Detection by RT-PCR of NRGs and erbB mRNAs in the medial basal hypothalamus of late juvenile 28- to 30-d-old female rats. M , DNA molecular size markers. NRG1, 268 bp; NRG2α, 314 bp; NRG2β, 270 bp; NRG3, 371 bp; erbB-2, 323 bp; erbB-3, 337 bp; and erbB-4, 168 bp. H , Hypothalamus; Hc , hippocampus; K , human keratinocytes; Lv , rat liver; bp , base pairs.

    Journal: The Journal of Neuroscience

    Article Title: Neuregulins Signaling via a Glial erbB-2–erbB-4 Receptor Complex Contribute to the Neuroendocrine Control of Mammalian Sexual Development

    doi: 10.1523/JNEUROSCI.19-22-09913.1999

    Figure Lengend Snippet: Detection by RT-PCR of NRGs and erbB mRNAs in the medial basal hypothalamus of late juvenile 28- to 30-d-old female rats. M , DNA molecular size markers. NRG1, 268 bp; NRG2α, 314 bp; NRG2β, 270 bp; NRG3, 371 bp; erbB-2, 323 bp; erbB-3, 337 bp; and erbB-4, 168 bp. H , Hypothalamus; Hc , hippocampus; K , human keratinocytes; Lv , rat liver; bp , base pairs.

    Article Snippet: Because the medial basal hypothalamic–median eminence region showed an abundance of erbB-2 and erbB-4 mRNA transcripts, we examined this region for the presence of erbB-2- and erbB-4-immunoreactive cells using immunohistofluorescence followed by confocal microscopy.

    Techniques: Reverse Transcription Polymerase Chain Reaction

    Delay of female puberty by targeted disruption of erbB-2 receptor synthesis via central administration of an erbB-2 ODN to developing rats. The erbB-ODN ( A ) or its scrambled sequence ( S ) were infused into the third ventricle of the brain via a stainless steel cannula connected to a subcutaneously implanted Alzet minipump delivering its content at the rate of 0.5 μl/hr for 14 d. The amount of ODN delivered was of 2.5 μg/hr. Numbers in parentheses are number of animals per group.

    Journal: The Journal of Neuroscience

    Article Title: Neuregulins Signaling via a Glial erbB-2–erbB-4 Receptor Complex Contribute to the Neuroendocrine Control of Mammalian Sexual Development

    doi: 10.1523/JNEUROSCI.19-22-09913.1999

    Figure Lengend Snippet: Delay of female puberty by targeted disruption of erbB-2 receptor synthesis via central administration of an erbB-2 ODN to developing rats. The erbB-ODN ( A ) or its scrambled sequence ( S ) were infused into the third ventricle of the brain via a stainless steel cannula connected to a subcutaneously implanted Alzet minipump delivering its content at the rate of 0.5 μl/hr for 14 d. The amount of ODN delivered was of 2.5 μg/hr. Numbers in parentheses are number of animals per group.

    Article Snippet: Because the medial basal hypothalamic–median eminence region showed an abundance of erbB-2 and erbB-4 mRNA transcripts, we examined this region for the presence of erbB-2- and erbB-4-immunoreactive cells using immunohistofluorescence followed by confocal microscopy.

    Techniques: Sequencing

    Cellular localization of erbB-4 and erbB-2 mRNAs in the brain of peripubertal female rats, as detected by in situ hybridization using 35 S-UTP-labeled cRNA probes. Within hypothalamic nuclei, the mRNA was found to be more abundant in cells of the paraventricular nuclei ( A , arrows ) and the ARC, VMH, and DMH nuclei ( B ). ErbB-4 mRNA is also diffusely detected throughout the medial basal hypothalamus ( B , C ) and is more abundantly present in cells of the subependymal region ( A , B , small arrowheads ) and glial cells in the intermediate and external layers of the median eminence ( B and C , small and large arrowheads , respectively). ErbB-2 transcripts were more abundantly expressed in ependymal cells lining the third ventricle ( E , F , arrowheads ) and glial cells of the median eminence ( F , arrows ). D and G depict sections adjacent to C and F hybridized with sense erbB-4 ( D ) and erbB-2 ( G ) RNA probes. Scale bars: B , 200 μm; A , C–G , 100 μm.

    Journal: The Journal of Neuroscience

    Article Title: Neuregulins Signaling via a Glial erbB-2–erbB-4 Receptor Complex Contribute to the Neuroendocrine Control of Mammalian Sexual Development

    doi: 10.1523/JNEUROSCI.19-22-09913.1999

    Figure Lengend Snippet: Cellular localization of erbB-4 and erbB-2 mRNAs in the brain of peripubertal female rats, as detected by in situ hybridization using 35 S-UTP-labeled cRNA probes. Within hypothalamic nuclei, the mRNA was found to be more abundant in cells of the paraventricular nuclei ( A , arrows ) and the ARC, VMH, and DMH nuclei ( B ). ErbB-4 mRNA is also diffusely detected throughout the medial basal hypothalamus ( B , C ) and is more abundantly present in cells of the subependymal region ( A , B , small arrowheads ) and glial cells in the intermediate and external layers of the median eminence ( B and C , small and large arrowheads , respectively). ErbB-2 transcripts were more abundantly expressed in ependymal cells lining the third ventricle ( E , F , arrowheads ) and glial cells of the median eminence ( F , arrows ). D and G depict sections adjacent to C and F hybridized with sense erbB-4 ( D ) and erbB-2 ( G ) RNA probes. Scale bars: B , 200 μm; A , C–G , 100 μm.

    Article Snippet: Because the medial basal hypothalamic–median eminence region showed an abundance of erbB-2 and erbB-4 mRNA transcripts, we examined this region for the presence of erbB-2- and erbB-4-immunoreactive cells using immunohistofluorescence followed by confocal microscopy.

    Techniques: In Situ Hybridization, Labeling

    Immunofluorescent confocal microcopy localization of erbB-2 in the medial basal hypothalamus–median eminence region of peripubertal female rats. ErbB-2 was visualized with a monoclonal antibody to an amino acid sequence contained in the human protein, and the reaction was developed with a second antibody conjugated to the fluorochrome Texas Red ( red ). Astrocytes were identified with a monoclonal antibody to GFAP directly labeled with the fluorochrome Oregon Green 488 ( green ). A, B , Low- and high-magnification views of the medial basal hypothalamus–median eminence region showing abundant erbB-2 immunoreactivity in tanycytes lining the wall of the third ventricle ( A , arrowheads ) and the processes that these cells send to the median eminence ( B , arrows ). Some neurons are also labeled ( A , B , short arrows ). C–E , Presence of erbB-2-immunoreactive material ( D , red ) in astrocytes ( C , green ) of the ventrolateral aspect of the median eminence (images merged in E ). F , Presence of erbB-2 in astrocytes adjacent to ependymal cells of the third ventricle. G–I , Higher magnification view of erbB-2-positive ( H ) astrocytes ( G ) adjacent to the third ventricle (images merged in I ). Notice the abundant erbB-2 immunoreactivity in GFAP-negative tanycytes. J–L , Detection of erbB-2 in astrocytes of the medial basal hypothalamus associated with blood vessels. Scale bars: A , 100 μm; B , 25 μm; C–E , G–L , 10 μm; F , 40 μm.

    Journal: The Journal of Neuroscience

    Article Title: Neuregulins Signaling via a Glial erbB-2–erbB-4 Receptor Complex Contribute to the Neuroendocrine Control of Mammalian Sexual Development

    doi: 10.1523/JNEUROSCI.19-22-09913.1999

    Figure Lengend Snippet: Immunofluorescent confocal microcopy localization of erbB-2 in the medial basal hypothalamus–median eminence region of peripubertal female rats. ErbB-2 was visualized with a monoclonal antibody to an amino acid sequence contained in the human protein, and the reaction was developed with a second antibody conjugated to the fluorochrome Texas Red ( red ). Astrocytes were identified with a monoclonal antibody to GFAP directly labeled with the fluorochrome Oregon Green 488 ( green ). A, B , Low- and high-magnification views of the medial basal hypothalamus–median eminence region showing abundant erbB-2 immunoreactivity in tanycytes lining the wall of the third ventricle ( A , arrowheads ) and the processes that these cells send to the median eminence ( B , arrows ). Some neurons are also labeled ( A , B , short arrows ). C–E , Presence of erbB-2-immunoreactive material ( D , red ) in astrocytes ( C , green ) of the ventrolateral aspect of the median eminence (images merged in E ). F , Presence of erbB-2 in astrocytes adjacent to ependymal cells of the third ventricle. G–I , Higher magnification view of erbB-2-positive ( H ) astrocytes ( G ) adjacent to the third ventricle (images merged in I ). Notice the abundant erbB-2 immunoreactivity in GFAP-negative tanycytes. J–L , Detection of erbB-2 in astrocytes of the medial basal hypothalamus associated with blood vessels. Scale bars: A , 100 μm; B , 25 μm; C–E , G–L , 10 μm; F , 40 μm.

    Article Snippet: Because the medial basal hypothalamic–median eminence region showed an abundance of erbB-2 and erbB-4 mRNA transcripts, we examined this region for the presence of erbB-2- and erbB-4-immunoreactive cells using immunohistofluorescence followed by confocal microscopy.

    Techniques: Sequencing, Labeling

    Increase in steady-state levels of erbB-2 and erbB-4 mRNA in the medial basal hypothalamus (ME-ARC region) of juvenile ovariectomized rats induced by E 2 or the sequential combination of E 2 plus P. The animals were ovariectomized on postnatal day 22 and received 5 d later a subcutaneous SILASTIC capsule containing 17β-E 2 at a concentration that produces preovulatory levels of serum E 2 . Control animals received a capsule filled with the vehicle ( V , corn oil). P was administered 50 hr later (at 12:00 P.M.) as a single subcutaneous injection. All animals were euthanized 4 hr after the P injection (i.e., at 4:00 P..M., 54 hr after receiving the E 2 -containing capsule). Each bar represents the mean ± SEM of three independent observations ( vertical lines ). Each observation derives from hypothalamic tissue pooled from three rats. ** p

    Journal: The Journal of Neuroscience

    Article Title: Neuregulins Signaling via a Glial erbB-2–erbB-4 Receptor Complex Contribute to the Neuroendocrine Control of Mammalian Sexual Development

    doi: 10.1523/JNEUROSCI.19-22-09913.1999

    Figure Lengend Snippet: Increase in steady-state levels of erbB-2 and erbB-4 mRNA in the medial basal hypothalamus (ME-ARC region) of juvenile ovariectomized rats induced by E 2 or the sequential combination of E 2 plus P. The animals were ovariectomized on postnatal day 22 and received 5 d later a subcutaneous SILASTIC capsule containing 17β-E 2 at a concentration that produces preovulatory levels of serum E 2 . Control animals received a capsule filled with the vehicle ( V , corn oil). P was administered 50 hr later (at 12:00 P.M.) as a single subcutaneous injection. All animals were euthanized 4 hr after the P injection (i.e., at 4:00 P..M., 54 hr after receiving the E 2 -containing capsule). Each bar represents the mean ± SEM of three independent observations ( vertical lines ). Each observation derives from hypothalamic tissue pooled from three rats. ** p

    Article Snippet: Because the medial basal hypothalamic–median eminence region showed an abundance of erbB-2 and erbB-4 mRNA transcripts, we examined this region for the presence of erbB-2- and erbB-4-immunoreactive cells using immunohistofluorescence followed by confocal microscopy.

    Techniques: Concentration Assay, Injection

    Regulation of MYCN expression by BMP and EGF signaling pathways. A , Wild type E11.5 embryonic hearts were cultured 2 hours with BMP10 (100 ng/ml), BMP inhibitor DMH1 (8 uM), Neuregulin-1 (625 ng/ml), or ERBB2 inhibitor AG1478 (100 uM). Western analysis

    Journal: Developmental biology

    Article Title: Myocardial Mycn is essential for mouse ventricular wall morphogenesis

    doi: 10.1016/j.ydbio.2012.10.005

    Figure Lengend Snippet: Regulation of MYCN expression by BMP and EGF signaling pathways. A , Wild type E11.5 embryonic hearts were cultured 2 hours with BMP10 (100 ng/ml), BMP inhibitor DMH1 (8 uM), Neuregulin-1 (625 ng/ml), or ERBB2 inhibitor AG1478 (100 uM). Western analysis

    Article Snippet: Embryonic hearts were treated with BMP10 (100ng/ml, R & D), BMP inhibitor DMH1 (8 uM, Sigma), Neuregulin-1 (625 ng/ml, R & D), or ERBB2 inhibitor AG1478 (100 uM, Calbiochem) for 22 hours before Western analysis.

    Techniques: Expressing, Cell Culture, Western Blot

    Relationship between Ets-1 52 kDa protein and HER-2/ neu (fmol mg −1 ). Ets-1 protein measurements were carried out using Western blotting. Arbitrary units were assigned to each protein band following scanning densitometry. Values are expressed as a ratio to β -actin. HER-2/ neu levels were measured using ELISA. Data was analysed using the nonparametric Spearman rank test.

    Journal: British Journal of Cancer

    Article Title: Overexpression of the Ets-1 transcription factor in human breast cancer

    doi: 10.1038/sj.bjc.6602128

    Figure Lengend Snippet: Relationship between Ets-1 52 kDa protein and HER-2/ neu (fmol mg −1 ). Ets-1 protein measurements were carried out using Western blotting. Arbitrary units were assigned to each protein band following scanning densitometry. Values are expressed as a ratio to β -actin. HER-2/ neu levels were measured using ELISA. Data was analysed using the nonparametric Spearman rank test.

    Article Snippet: Enzyme-linked immunosorbent assay (ELISA) uPA ELISA kits were obtained from American Diagnostica Inc, Greenwich, CT, USA; oestrogen receptor (ER) and progesterone receptor (PR) ELISA kits were obtained from Abbott Diagnostics, North Chicago, IL, USA, and the HER-2/neu ELISA kits were obtained from Oncogene Research products, Cambridge, MA, USA.

    Techniques: Western Blot, Enzyme-linked Immunosorbent Assay

    A : microRNA analysis of mmu-miR-296–3p, mmu-miR-200c*, and mmu-miR-465b-5p was differentially regulated by NRG1 and ErbB receptor inhibition. Cardiac differentiation of ESCs was performed by the hanging drop method. Cells were incubated with NRG1, ErbB2 inhibitor AG825 (1 μM), or ErbB1/ErbB2/ErbB4 inhibitor (1 nM) during day 5–7 . Cells were then incubated without stimulation. RNA was collected on day 9 . Accession number and name of the microRNAs are shown. B : mmu-miR-296–3p, mmu-miR-200c*, and mmu-miR-465b-5p were differentially expressed during hanging drop-induced mesoderm formation of ESCs. The expression of mmu-miR-296–3p and mmu-miR-200c* was increased during hanging drop-induced ESC differentiation. Expression of mmu-miR- 465b-5p was decreased during hanging drop-induced ESC differentiation. ESC differentiation was induced by the hanging drop method. RNA was collected on days 0 , 3, 5 , and 9 . Expression of mmu-miR-296–3p, mmu-miR-200c*, and mmu-miR-465b-5p was assessed by real-time PCR. Data were normalized to snoRNA202 and are presented as fold expression relative to the mRNA level of undifferentiated ESCs ( day 0 ). The results are from three independent experiments. * P

    Journal: American Journal of Physiology - Cell Physiology

    Article Title: Improving murine embryonic stem cell differentiation into cardiomyocytes with neuregulin-1: differential expression of microRNA

    doi: 10.1152/ajpcell.00141.2010

    Figure Lengend Snippet: A : microRNA analysis of mmu-miR-296–3p, mmu-miR-200c*, and mmu-miR-465b-5p was differentially regulated by NRG1 and ErbB receptor inhibition. Cardiac differentiation of ESCs was performed by the hanging drop method. Cells were incubated with NRG1, ErbB2 inhibitor AG825 (1 μM), or ErbB1/ErbB2/ErbB4 inhibitor (1 nM) during day 5–7 . Cells were then incubated without stimulation. RNA was collected on day 9 . Accession number and name of the microRNAs are shown. B : mmu-miR-296–3p, mmu-miR-200c*, and mmu-miR-465b-5p were differentially expressed during hanging drop-induced mesoderm formation of ESCs. The expression of mmu-miR-296–3p and mmu-miR-200c* was increased during hanging drop-induced ESC differentiation. Expression of mmu-miR- 465b-5p was decreased during hanging drop-induced ESC differentiation. ESC differentiation was induced by the hanging drop method. RNA was collected on days 0 , 3, 5 , and 9 . Expression of mmu-miR-296–3p, mmu-miR-200c*, and mmu-miR-465b-5p was assessed by real-time PCR. Data were normalized to snoRNA202 and are presented as fold expression relative to the mRNA level of undifferentiated ESCs ( day 0 ). The results are from three independent experiments. * P

    Article Snippet: NRG1 solvent (20 mM sodium acetate, 100 mM sodium sulfate, 1% mannitol, and 100 mM l -arginine, pH 6.5), recombinant human NRG1β [recombinant human glial growth factor 2 (rhGGF2), 100 ng/ml, a gift from Acorda Therapeutics], ErbB2 receptor inhibitor AG825 (1 μM, Calbiochem, San Diego, CA), or a ErbB1/ErbB2/ErbB4 receptor inhibitor (1 nM, catalog no. 324840, Calbiochem) was added in the culture medium at different time points.

    Techniques: Inhibition, Incubation, Expressing, Real-time Polymerase Chain Reaction

    Inhibition of ErbB2 or ErbB4 receptors inhibited hanging drop-induced cardiac differentiation of murine ESCs. A : inhibition of ErbB2 abolished NRG1-induced ErbB2 activation in ESCs. Six days after the initiation of hanging drop-induced ESC differentiation, cells were treated with NRG1 (100 ng/ml) for 2 h. An ErbB2 inhibitor (AG825, 1 μM) was added 1 h before NRG1 treatment. Total and phosphorylated ErbB receptor levels were measured by Western blot analysis. Densitometric quantification is shown at right . B : ErbB1/ErbB2/ErbB4 inhibitor abolished NRG1-induced activations of ErbB2 and ErbB4. Cells were treated and total and phosphorylated ErbB receptor levels were measured as described in A . Densitometric quantification is shown at right . C : inhibition of the ErbB2 and/or ErbB4 receptor decreased the protein level of NKX2.5 and the phosphorylation of Akt and ERK1/2. Cells were incubated with AG825 (1 μM) or an ErbB1/ErbB2/ErbB4 inhibitor (1 nM) during day 5–7 . Cells were then incubated without the inhibitor and collected on day 9 . NKX2.5, pAkt, and pERK1/2 were measured by Western blot analysis. GAPDH was used as loading control. D : inhibition of the ErbB2 and/or ErbB4 receptor decreased the mRNA of NKX2.5 and cTNT. mRNA expression of NKX2.5 and cTNT was assessed by real-time PCR. The results are from three independent experiments. * P

    Journal: American Journal of Physiology - Cell Physiology

    Article Title: Improving murine embryonic stem cell differentiation into cardiomyocytes with neuregulin-1: differential expression of microRNA

    doi: 10.1152/ajpcell.00141.2010

    Figure Lengend Snippet: Inhibition of ErbB2 or ErbB4 receptors inhibited hanging drop-induced cardiac differentiation of murine ESCs. A : inhibition of ErbB2 abolished NRG1-induced ErbB2 activation in ESCs. Six days after the initiation of hanging drop-induced ESC differentiation, cells were treated with NRG1 (100 ng/ml) for 2 h. An ErbB2 inhibitor (AG825, 1 μM) was added 1 h before NRG1 treatment. Total and phosphorylated ErbB receptor levels were measured by Western blot analysis. Densitometric quantification is shown at right . B : ErbB1/ErbB2/ErbB4 inhibitor abolished NRG1-induced activations of ErbB2 and ErbB4. Cells were treated and total and phosphorylated ErbB receptor levels were measured as described in A . Densitometric quantification is shown at right . C : inhibition of the ErbB2 and/or ErbB4 receptor decreased the protein level of NKX2.5 and the phosphorylation of Akt and ERK1/2. Cells were incubated with AG825 (1 μM) or an ErbB1/ErbB2/ErbB4 inhibitor (1 nM) during day 5–7 . Cells were then incubated without the inhibitor and collected on day 9 . NKX2.5, pAkt, and pERK1/2 were measured by Western blot analysis. GAPDH was used as loading control. D : inhibition of the ErbB2 and/or ErbB4 receptor decreased the mRNA of NKX2.5 and cTNT. mRNA expression of NKX2.5 and cTNT was assessed by real-time PCR. The results are from three independent experiments. * P

    Article Snippet: NRG1 solvent (20 mM sodium acetate, 100 mM sodium sulfate, 1% mannitol, and 100 mM l -arginine, pH 6.5), recombinant human NRG1β [recombinant human glial growth factor 2 (rhGGF2), 100 ng/ml, a gift from Acorda Therapeutics], ErbB2 receptor inhibitor AG825 (1 μM, Calbiochem, San Diego, CA), or a ErbB1/ErbB2/ErbB4 receptor inhibitor (1 nM, catalog no. 324840, Calbiochem) was added in the culture medium at different time points.

    Techniques: Inhibition, Activation Assay, Western Blot, Incubation, Expressing, Real-time Polymerase Chain Reaction

    Reverse phase protein analyses of ErbB2 + Ph + ALL with canertinib treatment. (A) Unsupervised clustering analyses were performed on RPPA data from Z181 or Z119 cells treated with 0.1–5 µM of canertinib for 18 hours. To generate heat maps, total protein and phosphoprotein levels were quantified by RPPA, and data were centered on the mean. Intensifying red color indicates increasing protein or phosphoprotein expression relative to the mean, black indicates the mean value, and intensifying green color indicates decreasing levels. (B) Supervised clustering showing relative changes in expression of pro-apoptotic proteins (Bim, cleaved PARP), protein kinase Cs, heat shock proteins, p38 mitogen activated protein kinase, and p38 T180/Y182p; five components of the phosphatidylinositol 3 kinase signaling pathway (Akt T308p, Akt S473p, p70S6-kinase T389p, S6-kinase S235/36p, and S6-kinase S240/44p); and ErbB2 Y1248p in the two cell lines after treatment.

    Journal: PLoS ONE

    Article Title: Small Molecule ErbB Inhibitors Decrease Proliferative Signaling and Promote Apoptosis in Philadelphia Chromosome-Positive Acute Lymphoblastic Leukemia

    doi: 10.1371/journal.pone.0070608

    Figure Lengend Snippet: Reverse phase protein analyses of ErbB2 + Ph + ALL with canertinib treatment. (A) Unsupervised clustering analyses were performed on RPPA data from Z181 or Z119 cells treated with 0.1–5 µM of canertinib for 18 hours. To generate heat maps, total protein and phosphoprotein levels were quantified by RPPA, and data were centered on the mean. Intensifying red color indicates increasing protein or phosphoprotein expression relative to the mean, black indicates the mean value, and intensifying green color indicates decreasing levels. (B) Supervised clustering showing relative changes in expression of pro-apoptotic proteins (Bim, cleaved PARP), protein kinase Cs, heat shock proteins, p38 mitogen activated protein kinase, and p38 T180/Y182p; five components of the phosphatidylinositol 3 kinase signaling pathway (Akt T308p, Akt S473p, p70S6-kinase T389p, S6-kinase S235/36p, and S6-kinase S240/44p); and ErbB2 Y1248p in the two cell lines after treatment.

    Article Snippet: Proteins were separated by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) and detected by Western Blot utilizing antibodies specific to ErbB2 (Cell Signaling Technology, Danvers, MA), ErbB2 Y1248p (Millipore, Billerica, MA), S6-kinase S240p (Cell Signaling Technology), S6-kinase (Cell Signaling Technology), p70S6-kinase (Epitomics, Burlingame, CA), p70S6-kinase T389p (Cell Signaling Technology), Bim (Epitomics), PARP (kindly provided by Dr. Scott Kaufman, Mayo Clinic, Rochester, MN), and actin (Sigma-Aldrich).

    Techniques: Expressing

    ErbB2 protein expression and activation in Ph + ALL cell lines. (A) Two established human Ph + ALL cell lines, Z181 and Z119, were lysed and then subjected to SDS-PAGE followed by Western blotting for ErbB2 and actin. Blots are representative of at least three independent experiments. Densitometry was performed using ImageJ (National Institutes of Health). (B) Cell lines were stained with murine PE-conjugated anti-human ErbB2 monoclonal antibody and assessed by flow cytometry; isotype control staining (grey) and anti-ErbB2 staining (white). Cell surface quantification was performed as described in Materials and Methods. Numbers indicate the average number of molecules of ErbB2 per cell. (C) Protein lysates were collected from cells treated with the indicated doses of canertinib for 18 hours. Samples were subjected to SDS-PAGE followed by Western blotting for ErbB2 Y1248p (ErbB2p), total ErbB2 (ErbB2), or actin. Blots are representative of at least three experiments. Densitometry was performed using ImageJ software and normalized to actin. (D) RPPA analyses were performed utilizing ErbB2p antibody. Bars represent the means of three individual experiments. Triangles indicate drug concentrations of 0–5 µM. *p

    Journal: PLoS ONE

    Article Title: Small Molecule ErbB Inhibitors Decrease Proliferative Signaling and Promote Apoptosis in Philadelphia Chromosome-Positive Acute Lymphoblastic Leukemia

    doi: 10.1371/journal.pone.0070608

    Figure Lengend Snippet: ErbB2 protein expression and activation in Ph + ALL cell lines. (A) Two established human Ph + ALL cell lines, Z181 and Z119, were lysed and then subjected to SDS-PAGE followed by Western blotting for ErbB2 and actin. Blots are representative of at least three independent experiments. Densitometry was performed using ImageJ (National Institutes of Health). (B) Cell lines were stained with murine PE-conjugated anti-human ErbB2 monoclonal antibody and assessed by flow cytometry; isotype control staining (grey) and anti-ErbB2 staining (white). Cell surface quantification was performed as described in Materials and Methods. Numbers indicate the average number of molecules of ErbB2 per cell. (C) Protein lysates were collected from cells treated with the indicated doses of canertinib for 18 hours. Samples were subjected to SDS-PAGE followed by Western blotting for ErbB2 Y1248p (ErbB2p), total ErbB2 (ErbB2), or actin. Blots are representative of at least three experiments. Densitometry was performed using ImageJ software and normalized to actin. (D) RPPA analyses were performed utilizing ErbB2p antibody. Bars represent the means of three individual experiments. Triangles indicate drug concentrations of 0–5 µM. *p

    Article Snippet: Proteins were separated by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) and detected by Western Blot utilizing antibodies specific to ErbB2 (Cell Signaling Technology, Danvers, MA), ErbB2 Y1248p (Millipore, Billerica, MA), S6-kinase S240p (Cell Signaling Technology), S6-kinase (Cell Signaling Technology), p70S6-kinase (Epitomics, Burlingame, CA), p70S6-kinase T389p (Cell Signaling Technology), Bim (Epitomics), PARP (kindly provided by Dr. Scott Kaufman, Mayo Clinic, Rochester, MN), and actin (Sigma-Aldrich).

    Techniques: Expressing, Activation Assay, SDS Page, Western Blot, Staining, Flow Cytometry, Cytometry, Software

    Msi1 'knockdown' by a shRNA preferentially reduces CD133 + cells and stem cell marker expression . A . Lentivirus expression of an Msi1 shRNA. Left, 'Knockdown' (KD) of Msi1 in MCF-7 and T47D spheroid cells by an Msi1 shRNA (Msi1 shRNA) reduces Msi1, CD133, ErbB2 and pERK expression vs. the control shRNA (Contr shRNA). Right panel, quantitation of the western blot indicates that Msi1 was reduced by 77-84% in MCF-7 and T47D cells. B . Msi1 KD reduces stem cell marker expression in MCF-7 and T47D cells. CD133, Nanog, Oct4, Sox2, c-Myc and Bmi1 mRNA levels were determined by qRT-PCR. All stem cell markers, with the exception of Oct4 in T47D cells, were reduced by Msi1 knockdown. C . Left panel, Msi1 KD reduces Notch1 and increases p21 CIP1 in MCF-7 and T47D spheroid cells. IC, Notch intracellular domain; FL, full-length Notch1. Middle panel, quantitation of Notch1 expression. Right panel, quantitation of p21 CIP1 expression. D . Msi1 KD reduces Notch1 mRNA 2.7-5-fold, but not p21 CIP1 , in MCF-7 and T47D spheroid cells, respectively.

    Journal: Molecular Cancer

    Article Title: Musashi1 regulates breast tumor cell proliferation and is a prognostic indicator of poor survival

    doi: 10.1186/1476-4598-9-221

    Figure Lengend Snippet: Msi1 'knockdown' by a shRNA preferentially reduces CD133 + cells and stem cell marker expression . A . Lentivirus expression of an Msi1 shRNA. Left, 'Knockdown' (KD) of Msi1 in MCF-7 and T47D spheroid cells by an Msi1 shRNA (Msi1 shRNA) reduces Msi1, CD133, ErbB2 and pERK expression vs. the control shRNA (Contr shRNA). Right panel, quantitation of the western blot indicates that Msi1 was reduced by 77-84% in MCF-7 and T47D cells. B . Msi1 KD reduces stem cell marker expression in MCF-7 and T47D cells. CD133, Nanog, Oct4, Sox2, c-Myc and Bmi1 mRNA levels were determined by qRT-PCR. All stem cell markers, with the exception of Oct4 in T47D cells, were reduced by Msi1 knockdown. C . Left panel, Msi1 KD reduces Notch1 and increases p21 CIP1 in MCF-7 and T47D spheroid cells. IC, Notch intracellular domain; FL, full-length Notch1. Middle panel, quantitation of Notch1 expression. Right panel, quantitation of p21 CIP1 expression. D . Msi1 KD reduces Notch1 mRNA 2.7-5-fold, but not p21 CIP1 , in MCF-7 and T47D spheroid cells, respectively.

    Article Snippet: Primary antibodies included rat anti-Msi1 or anti-biotinylated Msi1 (1:1000) [ ] (14H-1, Dr. Hideyuki Okano, Keio University, Tokyo, Japan), rabbit anti-ErbB2 (1:1000) (Millipore, Danvers, MA), mouse anti-ERα (1:200, Santa Cruz Biotechnology, Santa Cruz, CA), rabbit anti-CD133 (1:1000) (Cell Signaling Technology, Danvers, MA), rabbit anti-pERK1/2 (1:1000)(Cell Signaling Technology), mouse anti-β-actin (1:5000) (Sigma-Aldrich Corp), rabbit anti-Notch1(D1E11) (Cell Signaling Technology) and mouse anti-p21CIP1 (EMD Chemicals, Gibbstown, NJ).

    Techniques: shRNA, Marker, Expressing, Quantitation Assay, Western Blot, Quantitative RT-PCR

    Msi1 correlates with ErbB2 expression . A . Msi1, ErbB2, ERα and β-actin were measured in 20 breast cancer cell lines and in immortalized breast epithelial cell line MCF-10A by western blotting. B . There is a statistically significant correlation between Msi1 and ErbB2 (P = 0.02) by Fisher's Exact test.

    Journal: Molecular Cancer

    Article Title: Musashi1 regulates breast tumor cell proliferation and is a prognostic indicator of poor survival

    doi: 10.1186/1476-4598-9-221

    Figure Lengend Snippet: Msi1 correlates with ErbB2 expression . A . Msi1, ErbB2, ERα and β-actin were measured in 20 breast cancer cell lines and in immortalized breast epithelial cell line MCF-10A by western blotting. B . There is a statistically significant correlation between Msi1 and ErbB2 (P = 0.02) by Fisher's Exact test.

    Article Snippet: Primary antibodies included rat anti-Msi1 or anti-biotinylated Msi1 (1:1000) [ ] (14H-1, Dr. Hideyuki Okano, Keio University, Tokyo, Japan), rabbit anti-ErbB2 (1:1000) (Millipore, Danvers, MA), mouse anti-ERα (1:200, Santa Cruz Biotechnology, Santa Cruz, CA), rabbit anti-CD133 (1:1000) (Cell Signaling Technology, Danvers, MA), rabbit anti-pERK1/2 (1:1000)(Cell Signaling Technology), mouse anti-β-actin (1:5000) (Sigma-Aldrich Corp), rabbit anti-Notch1(D1E11) (Cell Signaling Technology) and mouse anti-p21CIP1 (EMD Chemicals, Gibbstown, NJ).

    Techniques: Expressing, Western Blot

    IbTX differentially regulates oncogenic pathways. (A) IbTX attenuated the HER-2/neu levels in the UACC893 cells ( HER-2/neu , UACC893 ). SK-BR-3 cells express full length ( HER-2/neu , SK-BR-3 ) and truncated ( p95 HER-2/neu , SK-BR-3 ) oncoprotein isoforms with both being increased by IbTX. (B) IbTX downregulated the full length ( β-catenin ) but not truncated ( trβ-catenin ) β-catenin isoforms in the UACC893 and MDA-MB-231 cells. (C) In the UACC893 cells, IbTX attenuated phosphorylated ( T308pAkt , UACC893 ) but not total Akt ( Akt , UACC893 ). Both phosphorylated ( T308pAkt , MDA-MB-231 ) and total ( Akt , MDA-MB-231 ) Akt were decreased in the MDA-MB-231 cells with IbTX. In SK-BR-3 cells, phosphorylated ( T308pAkt , SK-BR-3 ) and total ( Akt , SK-BR-3 ) Akt increased at high IbTX concentrations. (D) GAPDH was used in the UACC893, MDA-MB-231 and SK-BR-3 cell immunoblotting to ensure equal protein loading. IbTX, iberiotoxin.

    Journal: Oncology Reports

    Article Title: BKCa channel inhibitor modulates the tumorigenic ability of hormone-independent breast cancer cells via the Wnt pathway

    doi: 10.3892/or.2014.3617

    Figure Lengend Snippet: IbTX differentially regulates oncogenic pathways. (A) IbTX attenuated the HER-2/neu levels in the UACC893 cells ( HER-2/neu , UACC893 ). SK-BR-3 cells express full length ( HER-2/neu , SK-BR-3 ) and truncated ( p95 HER-2/neu , SK-BR-3 ) oncoprotein isoforms with both being increased by IbTX. (B) IbTX downregulated the full length ( β-catenin ) but not truncated ( trβ-catenin ) β-catenin isoforms in the UACC893 and MDA-MB-231 cells. (C) In the UACC893 cells, IbTX attenuated phosphorylated ( T308pAkt , UACC893 ) but not total Akt ( Akt , UACC893 ). Both phosphorylated ( T308pAkt , MDA-MB-231 ) and total ( Akt , MDA-MB-231 ) Akt were decreased in the MDA-MB-231 cells with IbTX. In SK-BR-3 cells, phosphorylated ( T308pAkt , SK-BR-3 ) and total ( Akt , SK-BR-3 ) Akt increased at high IbTX concentrations. (D) GAPDH was used in the UACC893, MDA-MB-231 and SK-BR-3 cell immunoblotting to ensure equal protein loading. IbTX, iberiotoxin.

    Article Snippet: Blots were then incubated with the rabbit anti-HER-2/neu antibody (1:500 EMD; Millipore, Billerica, MA, USA), rabbit β-catenin antibody (1:500), rabbit T308 phospho-Akt or total Akt antibody (both from Cell Signaling Technology, Danvers, MA, USA) overnight at 4°C.

    Techniques: Multiple Displacement Amplification

    In BaF3 ErbB2/ErbB4 cells, anti-ErbB2 and anti-ErbB4 antibodies do not cross-react with the noncognate ErbB receptor. BaF3 ErbB2/ErbB4 cells were starved and stimulated with NRG2β. ErbB2 or ErbB4 was immunoprecipitated using ErbB2- and ErbB4-specific

    Journal: Genes & Cancer

    Article Title: ErbB2 Is Necessary for ErbB4 Ligands to Stimulate Oncogenic Activities in Models of Human Breast Cancer

    doi: 10.1177/1947601911431080

    Figure Lengend Snippet: In BaF3 ErbB2/ErbB4 cells, anti-ErbB2 and anti-ErbB4 antibodies do not cross-react with the noncognate ErbB receptor. BaF3 ErbB2/ErbB4 cells were starved and stimulated with NRG2β. ErbB2 or ErbB4 was immunoprecipitated using ErbB2- and ErbB4-specific

    Article Snippet: ErbB receptor expression and tyrosine phosphorylation were assayed by immunoprecipitation and immunoblotting essentially as described., , Briefly, ErbB2 was precipitated using an anti-ErbB2 mouse monoclonal antibody (OP-39; EMD Chemicals, Inc., Gibbstown, NJ); ErbB4 was precipitated using an anti-ErbB4 mouse monoclonal antibody (SC-8050; Santa Cruz Biotechnology, Santa Cruz, CA).

    Techniques: Immunoprecipitation

    Silencing expression of ErbB4 or ErbB2 reduces NRG2β stimulation of migration in the MCF7 human breast tumor cell line

    Journal: Genes & Cancer

    Article Title: ErbB2 Is Necessary for ErbB4 Ligands to Stimulate Oncogenic Activities in Models of Human Breast Cancer

    doi: 10.1177/1947601911431080

    Figure Lengend Snippet: Silencing expression of ErbB4 or ErbB2 reduces NRG2β stimulation of migration in the MCF7 human breast tumor cell line

    Article Snippet: ErbB receptor expression and tyrosine phosphorylation were assayed by immunoprecipitation and immunoblotting essentially as described., , Briefly, ErbB2 was precipitated using an anti-ErbB2 mouse monoclonal antibody (OP-39; EMD Chemicals, Inc., Gibbstown, NJ); ErbB4 was precipitated using an anti-ErbB4 mouse monoclonal antibody (SC-8050; Santa Cruz Biotechnology, Santa Cruz, CA).

    Techniques: Expressing, Migration

    Crosstalk between ErbB4 and ErbB2 may account for ErbB4 coupling to cell proliferation. We propose that ErbB4/ErbB2 crosstalk occurs through ErbB4 phosphorylation by ErbB2.

    Journal: Genes & Cancer

    Article Title: ErbB2 Is Necessary for ErbB4 Ligands to Stimulate Oncogenic Activities in Models of Human Breast Cancer

    doi: 10.1177/1947601911431080

    Figure Lengend Snippet: Crosstalk between ErbB4 and ErbB2 may account for ErbB4 coupling to cell proliferation. We propose that ErbB4/ErbB2 crosstalk occurs through ErbB4 phosphorylation by ErbB2.

    Article Snippet: ErbB receptor expression and tyrosine phosphorylation were assayed by immunoprecipitation and immunoblotting essentially as described., , Briefly, ErbB2 was precipitated using an anti-ErbB2 mouse monoclonal antibody (OP-39; EMD Chemicals, Inc., Gibbstown, NJ); ErbB4 was precipitated using an anti-ErbB4 mouse monoclonal antibody (SC-8050; Santa Cruz Biotechnology, Santa Cruz, CA).

    Techniques:

    Silencing endogenous ErbB4 or ErbB2 transcription in the MCF7 human breast tumor cell line reduces stimulation of motility by NRG2β. ( a ) ErbB2 expression ( upper panel ) and ErbB4 expression ( lower panel ) were evaluated in MCF7 cell lines that express

    Journal: Genes & Cancer

    Article Title: ErbB2 Is Necessary for ErbB4 Ligands to Stimulate Oncogenic Activities in Models of Human Breast Cancer

    doi: 10.1177/1947601911431080

    Figure Lengend Snippet: Silencing endogenous ErbB4 or ErbB2 transcription in the MCF7 human breast tumor cell line reduces stimulation of motility by NRG2β. ( a ) ErbB2 expression ( upper panel ) and ErbB4 expression ( lower panel ) were evaluated in MCF7 cell lines that express

    Article Snippet: ErbB receptor expression and tyrosine phosphorylation were assayed by immunoprecipitation and immunoblotting essentially as described., , Briefly, ErbB2 was precipitated using an anti-ErbB2 mouse monoclonal antibody (OP-39; EMD Chemicals, Inc., Gibbstown, NJ); ErbB4 was precipitated using an anti-ErbB4 mouse monoclonal antibody (SC-8050; Santa Cruz Biotechnology, Santa Cruz, CA).

    Techniques: Expressing

    ErbB2 kinase activity and sites of ErbB4 tyrosine phosphorylation are required for ligand-induced heterotypic ErbB4 signaling

    Journal: Genes & Cancer

    Article Title: ErbB2 Is Necessary for ErbB4 Ligands to Stimulate Oncogenic Activities in Models of Human Breast Cancer

    doi: 10.1177/1947601911431080

    Figure Lengend Snippet: ErbB2 kinase activity and sites of ErbB4 tyrosine phosphorylation are required for ligand-induced heterotypic ErbB4 signaling

    Article Snippet: ErbB receptor expression and tyrosine phosphorylation were assayed by immunoprecipitation and immunoblotting essentially as described., , Briefly, ErbB2 was precipitated using an anti-ErbB2 mouse monoclonal antibody (OP-39; EMD Chemicals, Inc., Gibbstown, NJ); ErbB4 was precipitated using an anti-ErbB4 mouse monoclonal antibody (SC-8050; Santa Cruz Biotechnology, Santa Cruz, CA).

    Techniques: Activity Assay

    In BaF3 ErbB2/ErbB4 cells, ErbB4 tyrosine phosphorylation is abrogated by mutating 9 ErbB4 cytoplasmic tyrosine residues but not by disrupting ErbB4 kinase activity. BaF3 ErbB2/ErbB4 cell lines were starved and stimulated with NRG2β. Immunoprecipitation

    Journal: Genes & Cancer

    Article Title: ErbB2 Is Necessary for ErbB4 Ligands to Stimulate Oncogenic Activities in Models of Human Breast Cancer

    doi: 10.1177/1947601911431080

    Figure Lengend Snippet: In BaF3 ErbB2/ErbB4 cells, ErbB4 tyrosine phosphorylation is abrogated by mutating 9 ErbB4 cytoplasmic tyrosine residues but not by disrupting ErbB4 kinase activity. BaF3 ErbB2/ErbB4 cell lines were starved and stimulated with NRG2β. Immunoprecipitation

    Article Snippet: ErbB receptor expression and tyrosine phosphorylation were assayed by immunoprecipitation and immunoblotting essentially as described., , Briefly, ErbB2 was precipitated using an anti-ErbB2 mouse monoclonal antibody (OP-39; EMD Chemicals, Inc., Gibbstown, NJ); ErbB4 was precipitated using an anti-ErbB4 mouse monoclonal antibody (SC-8050; Santa Cruz Biotechnology, Santa Cruz, CA).

    Techniques: Activity Assay, Immunoprecipitation

    Silencing endogenous ErbB4 or ErbB2 transcription in the T47D human breast tumor cell line reduces stimulation of anchorage-independent proliferation by NRG2β. ( a ) ErbB2 expression ( upper panel ) and ErbB4 expression ( lower panel ) were evaluated

    Journal: Genes & Cancer

    Article Title: ErbB2 Is Necessary for ErbB4 Ligands to Stimulate Oncogenic Activities in Models of Human Breast Cancer

    doi: 10.1177/1947601911431080

    Figure Lengend Snippet: Silencing endogenous ErbB4 or ErbB2 transcription in the T47D human breast tumor cell line reduces stimulation of anchorage-independent proliferation by NRG2β. ( a ) ErbB2 expression ( upper panel ) and ErbB4 expression ( lower panel ) were evaluated

    Article Snippet: ErbB receptor expression and tyrosine phosphorylation were assayed by immunoprecipitation and immunoblotting essentially as described., , Briefly, ErbB2 was precipitated using an anti-ErbB2 mouse monoclonal antibody (OP-39; EMD Chemicals, Inc., Gibbstown, NJ); ErbB4 was precipitated using an anti-ErbB4 mouse monoclonal antibody (SC-8050; Santa Cruz Biotechnology, Santa Cruz, CA).

    Techniques: Expressing

    Silencing expression of ErbB4 or ErbB2 reduces NRG2β stimulation of anchorage-independent proliferation in the T47D human breast tumor cell line

    Journal: Genes & Cancer

    Article Title: ErbB2 Is Necessary for ErbB4 Ligands to Stimulate Oncogenic Activities in Models of Human Breast Cancer

    doi: 10.1177/1947601911431080

    Figure Lengend Snippet: Silencing expression of ErbB4 or ErbB2 reduces NRG2β stimulation of anchorage-independent proliferation in the T47D human breast tumor cell line

    Article Snippet: ErbB receptor expression and tyrosine phosphorylation were assayed by immunoprecipitation and immunoblotting essentially as described., , Briefly, ErbB2 was precipitated using an anti-ErbB2 mouse monoclonal antibody (OP-39; EMD Chemicals, Inc., Gibbstown, NJ); ErbB4 was precipitated using an anti-ErbB4 mouse monoclonal antibody (SC-8050; Santa Cruz Biotechnology, Santa Cruz, CA).

    Techniques: Expressing

    Lrig1 controls endogenous signalling via the ErbB pathway a) Loss of Lrig1 causes increased protein levels and activation of the ErbB pathway. pEgfr, Egfr, pErbB2, ErbB2, pErbB3, ErbB3, cMet were detected by Western blotting in samples enriched for intestinal epithelium. β-actin is used a loading control. b) Relative expression analysis of the receptors by qPCR at P10 shows minor differences. Expression levels are shown relative to control samples (KO/Ctrl). Asterisks indicate significant changes ( ErbB2 : p=0.004; ErbB3 : p=0.04; KO n=4, Ctrl n=3). c-f) Increased activation of MAPK signalling and cMyc signalling upon loss of Lrig1. Immunohistochemical analysis for p-MEK1-2 (c-d) and cMyc (e-f). g-i) Altered Egfr activation dynamics upon loss of Lrig1 KO. i) Average normalised membrane intensity of pEgfr in intestinal samples from KO (n=6) and control animals (n=10) for 6-18 individual crypts per sample. Error bars represent the s.e.m. (positions 1-4: p

    Journal: Nature cell biology

    Article Title: Lrig1 controls intestinal stem cell homeostasis by negative regulation of ErbB signalling

    doi: 10.1038/ncb2464

    Figure Lengend Snippet: Lrig1 controls endogenous signalling via the ErbB pathway a) Loss of Lrig1 causes increased protein levels and activation of the ErbB pathway. pEgfr, Egfr, pErbB2, ErbB2, pErbB3, ErbB3, cMet were detected by Western blotting in samples enriched for intestinal epithelium. β-actin is used a loading control. b) Relative expression analysis of the receptors by qPCR at P10 shows minor differences. Expression levels are shown relative to control samples (KO/Ctrl). Asterisks indicate significant changes ( ErbB2 : p=0.004; ErbB3 : p=0.04; KO n=4, Ctrl n=3). c-f) Increased activation of MAPK signalling and cMyc signalling upon loss of Lrig1. Immunohistochemical analysis for p-MEK1-2 (c-d) and cMyc (e-f). g-i) Altered Egfr activation dynamics upon loss of Lrig1 KO. i) Average normalised membrane intensity of pEgfr in intestinal samples from KO (n=6) and control animals (n=10) for 6-18 individual crypts per sample. Error bars represent the s.e.m. (positions 1-4: p

    Article Snippet: Antibodies The following antibodies were used rabbit anti-β-catenin (Santa Cruz, sc-7199, 1:250), rabbit anti-Phospho-histone H3 (ser10) (Cell Signaling #9701, 1:400), rabbit anti-phospho-MEK1/2 (Ser221) (Cell signalling, #2338, 1:500), mouse anti-BrdU (Cell signaling, Bu20a, 1:250), goat anti-Lrig1(R and D Systems, AF3688, 1:100), rabbit anti-Myc (Millipore, 06-340, 1:100), mouse anti-Ki67 (Monosan, Clone MM1, 1:1000), PE-conjugated anti-Lrig1 (R and D systems, FAB3688P, 10μL/106 cells), Pacific blue and Alexa647 conjugated Rat anti-CD24 (Biolegend, M1/69, 5μL/106 cells), rat anti-Msi1 (MBL, D270-6, 1:10000), rabbit anti-phospho Egfr (Y1068) (Abcam, EP7774Y, 1:250), rabbit anti-Egfr (Cell Signaling, #4267 clone D38B1, 1:250), rabbit anti-phospho ErbB2 (Y1248) (Abcam, ab47755), mouse anti-ErbB2 (Millipore, 05-1130, Clone N3D10, 1:500), rabbit anti-phospho ErbB3 (Y1289) (Cell Signaling, #4791, Clone 21D3, 1:250), mouse anti-ErbB3 (Millipore, 05-390, Clone 2F12, 1:500), rabbit anti-cMet (Santa Cruz, sc-162, 1:500), mouse anti-β-actin, (Sigma A5316, clone AC-74, 1:5000) and Atto-488 conjugated UEA-1 (Sigma, 10μL/106 cells for flow cytometry, 1:1000 for near-native sections) according to manufacturer’s instructions.

    Techniques: Activation Assay, Western Blot, Expressing, Real-time Polymerase Chain Reaction, Immunohistochemistry

    HK II dissociation from mitochondria is increased in ErbB2-overexpressing breast cancer cells. (A) The intact mitochondrial fractions from 231V and 231ErbB2 cells and (B) BT474 scramble and BT474 siErbB2 cells were isolated and treated with 3-BrPA for

    Journal: Oncology Letters

    Article Title: Overexpression of ErbB2 renders breast cancer cells susceptible to 3-BrPA through the increased dissociation of hexokinase II from mitochondrial outer membrane

    doi: 10.3892/ol.2015.4043

    Figure Lengend Snippet: HK II dissociation from mitochondria is increased in ErbB2-overexpressing breast cancer cells. (A) The intact mitochondrial fractions from 231V and 231ErbB2 cells and (B) BT474 scramble and BT474 siErbB2 cells were isolated and treated with 3-BrPA for

    Article Snippet: The following antibodies were utilized: anti-ErbB2 mouse antibody (1:1000, OP15, Calbiochem, National Harbor, MD, USA), anti-HK II mouse antibody (1:1000, no. 8169, Cell Signaling Technology Inc., Beverly, MA, USA), anti-prohibitin rabbit antibody (1:1000, no. 2426, Cell Signaling Technology Inc.), anti-α-Tubulin rabbit antibody (1:1000, no. 2125, Cell Signaling Technology Inc.), anti-mtHSP70 mouse antibody (1:1000, MA3-028, Thermo Scientific, Carlsbad, CA, USA), anti-cytochrome C rabbit antibody (1:1000, no. 11940, Cell Signaling Technology Inc.), anti-PARP rabbit antibody (1:1000, no. 9542, Cell Signaling Technology Inc.), anti-AIF rabbit antibody (1:1000, no. 4642, Cell Signaling Technology Inc.) and anti-β-actin rabbit antibody (1:2000, no. 4970, Cell Signaling Technology Inc.).

    Techniques: Isolation

    Overexpression of ErbB2 in breast cancer cells increases mitochondrial localization and activity of HK II. (A) Overexpression of ErbB2 upregulated the expression of HK II. Whole cell lysates were collected and for western blotting analysis, and β-actin

    Journal: Oncology Letters

    Article Title: Overexpression of ErbB2 renders breast cancer cells susceptible to 3-BrPA through the increased dissociation of hexokinase II from mitochondrial outer membrane

    doi: 10.3892/ol.2015.4043

    Figure Lengend Snippet: Overexpression of ErbB2 in breast cancer cells increases mitochondrial localization and activity of HK II. (A) Overexpression of ErbB2 upregulated the expression of HK II. Whole cell lysates were collected and for western blotting analysis, and β-actin

    Article Snippet: The following antibodies were utilized: anti-ErbB2 mouse antibody (1:1000, OP15, Calbiochem, National Harbor, MD, USA), anti-HK II mouse antibody (1:1000, no. 8169, Cell Signaling Technology Inc., Beverly, MA, USA), anti-prohibitin rabbit antibody (1:1000, no. 2426, Cell Signaling Technology Inc.), anti-α-Tubulin rabbit antibody (1:1000, no. 2125, Cell Signaling Technology Inc.), anti-mtHSP70 mouse antibody (1:1000, MA3-028, Thermo Scientific, Carlsbad, CA, USA), anti-cytochrome C rabbit antibody (1:1000, no. 11940, Cell Signaling Technology Inc.), anti-PARP rabbit antibody (1:1000, no. 9542, Cell Signaling Technology Inc.), anti-AIF rabbit antibody (1:1000, no. 4642, Cell Signaling Technology Inc.) and anti-β-actin rabbit antibody (1:2000, no. 4970, Cell Signaling Technology Inc.).

    Techniques: Over Expression, Activity Assay, Expressing, Western Blot

    3-BrPA has a greater inhibitory effect in ErbB2-overexpressing breast cancer cells than control cells in nude mice. Pre-established 231V or 231ErbB2 tumor xenografts were treated with control (phosphate-buffered saline), 3-BrPA (10 mg/kg, twice/week for

    Journal: Oncology Letters

    Article Title: Overexpression of ErbB2 renders breast cancer cells susceptible to 3-BrPA through the increased dissociation of hexokinase II from mitochondrial outer membrane

    doi: 10.3892/ol.2015.4043

    Figure Lengend Snippet: 3-BrPA has a greater inhibitory effect in ErbB2-overexpressing breast cancer cells than control cells in nude mice. Pre-established 231V or 231ErbB2 tumor xenografts were treated with control (phosphate-buffered saline), 3-BrPA (10 mg/kg, twice/week for

    Article Snippet: The following antibodies were utilized: anti-ErbB2 mouse antibody (1:1000, OP15, Calbiochem, National Harbor, MD, USA), anti-HK II mouse antibody (1:1000, no. 8169, Cell Signaling Technology Inc., Beverly, MA, USA), anti-prohibitin rabbit antibody (1:1000, no. 2426, Cell Signaling Technology Inc.), anti-α-Tubulin rabbit antibody (1:1000, no. 2125, Cell Signaling Technology Inc.), anti-mtHSP70 mouse antibody (1:1000, MA3-028, Thermo Scientific, Carlsbad, CA, USA), anti-cytochrome C rabbit antibody (1:1000, no. 11940, Cell Signaling Technology Inc.), anti-PARP rabbit antibody (1:1000, no. 9542, Cell Signaling Technology Inc.), anti-AIF rabbit antibody (1:1000, no. 4642, Cell Signaling Technology Inc.) and anti-β-actin rabbit antibody (1:2000, no. 4970, Cell Signaling Technology Inc.).

    Techniques: Mouse Assay

    Breast cancer cells with a high expression of ErbB2 are more sensitive to glucose depletion than ErbB2-negative cells. (A) Overexpression of ErbB2 in MCF7 and MDA-MB-231 breast cancer cells, and siRNA knockdown of ErbB2 in BT474 breast cancer cells. Cells

    Journal: Oncology Letters

    Article Title: Overexpression of ErbB2 renders breast cancer cells susceptible to 3-BrPA through the increased dissociation of hexokinase II from mitochondrial outer membrane

    doi: 10.3892/ol.2015.4043

    Figure Lengend Snippet: Breast cancer cells with a high expression of ErbB2 are more sensitive to glucose depletion than ErbB2-negative cells. (A) Overexpression of ErbB2 in MCF7 and MDA-MB-231 breast cancer cells, and siRNA knockdown of ErbB2 in BT474 breast cancer cells. Cells

    Article Snippet: The following antibodies were utilized: anti-ErbB2 mouse antibody (1:1000, OP15, Calbiochem, National Harbor, MD, USA), anti-HK II mouse antibody (1:1000, no. 8169, Cell Signaling Technology Inc., Beverly, MA, USA), anti-prohibitin rabbit antibody (1:1000, no. 2426, Cell Signaling Technology Inc.), anti-α-Tubulin rabbit antibody (1:1000, no. 2125, Cell Signaling Technology Inc.), anti-mtHSP70 mouse antibody (1:1000, MA3-028, Thermo Scientific, Carlsbad, CA, USA), anti-cytochrome C rabbit antibody (1:1000, no. 11940, Cell Signaling Technology Inc.), anti-PARP rabbit antibody (1:1000, no. 9542, Cell Signaling Technology Inc.), anti-AIF rabbit antibody (1:1000, no. 4642, Cell Signaling Technology Inc.) and anti-β-actin rabbit antibody (1:2000, no. 4970, Cell Signaling Technology Inc.).

    Techniques: Expressing, Over Expression, Multiple Displacement Amplification

    Overexpression of ErbB2 decreased the mitochondria membrane potential following 3-BrPA treatment, inducing apoptosis. (A) The mitochondria membrane potential (MMP) was measured in 231V, 231ErbB2, BT474 scramble and BT474 siErbB2 cells following treatment

    Journal: Oncology Letters

    Article Title: Overexpression of ErbB2 renders breast cancer cells susceptible to 3-BrPA through the increased dissociation of hexokinase II from mitochondrial outer membrane

    doi: 10.3892/ol.2015.4043

    Figure Lengend Snippet: Overexpression of ErbB2 decreased the mitochondria membrane potential following 3-BrPA treatment, inducing apoptosis. (A) The mitochondria membrane potential (MMP) was measured in 231V, 231ErbB2, BT474 scramble and BT474 siErbB2 cells following treatment

    Article Snippet: The following antibodies were utilized: anti-ErbB2 mouse antibody (1:1000, OP15, Calbiochem, National Harbor, MD, USA), anti-HK II mouse antibody (1:1000, no. 8169, Cell Signaling Technology Inc., Beverly, MA, USA), anti-prohibitin rabbit antibody (1:1000, no. 2426, Cell Signaling Technology Inc.), anti-α-Tubulin rabbit antibody (1:1000, no. 2125, Cell Signaling Technology Inc.), anti-mtHSP70 mouse antibody (1:1000, MA3-028, Thermo Scientific, Carlsbad, CA, USA), anti-cytochrome C rabbit antibody (1:1000, no. 11940, Cell Signaling Technology Inc.), anti-PARP rabbit antibody (1:1000, no. 9542, Cell Signaling Technology Inc.), anti-AIF rabbit antibody (1:1000, no. 4642, Cell Signaling Technology Inc.) and anti-β-actin rabbit antibody (1:2000, no. 4970, Cell Signaling Technology Inc.).

    Techniques: Over Expression

    ErbB2 internalization and lysosomal trafficking induced by proteasome inhibition is clathrin-independent and requires new protein synthesis. A. Immunofluorescence imaging of control and PS341 treated (10nM, 24h) SK-BR-3 cells showing redistribution of

    Journal: Cancer research

    Article Title: ErbB2 Trafficking and Degradation Associated with K48 and K63 Polyubiquitination

    doi: 10.1158/0008-5472.CAN-09-3768

    Figure Lengend Snippet: ErbB2 internalization and lysosomal trafficking induced by proteasome inhibition is clathrin-independent and requires new protein synthesis. A. Immunofluorescence imaging of control and PS341 treated (10nM, 24h) SK-BR-3 cells showing redistribution of

    Article Snippet: The mouse IgG1 monoclonal anti-c-ErbB2/c-Neu (Ab-3), developed against the C-terminal 1242–1255 amino acids of human ErbB2, was purchased from Calbiochem (San Diego, CA).

    Techniques: Inhibition, Immunofluorescence, Imaging

    Proteasome and HSP90 inhibition induce differential rates of ErbB2 chaperone exchange, internalization and decay in SK-BR-3 cells. A. Immunoblots (IB) of whole cell extracts showing loss of 185 kDa ErbB2 protein expression (above), and ErbB2 immunoprecipitates

    Journal: Cancer research

    Article Title: ErbB2 Trafficking and Degradation Associated with K48 and K63 Polyubiquitination

    doi: 10.1158/0008-5472.CAN-09-3768

    Figure Lengend Snippet: Proteasome and HSP90 inhibition induce differential rates of ErbB2 chaperone exchange, internalization and decay in SK-BR-3 cells. A. Immunoblots (IB) of whole cell extracts showing loss of 185 kDa ErbB2 protein expression (above), and ErbB2 immunoprecipitates

    Article Snippet: The mouse IgG1 monoclonal anti-c-ErbB2/c-Neu (Ab-3), developed against the C-terminal 1242–1255 amino acids of human ErbB2, was purchased from Calbiochem (San Diego, CA).

    Techniques: Inhibition, Western Blot, Expressing

    ErbB2 receptor overexpression in SK-BR-3 is regulated by both polyubiquitinating and deubiquitinating mechanisms. A. Immunofluorescence images of ErbB2 expression in SK-BR-3 cells transiently transfected with constructs overexpressing either wildtype

    Journal: Cancer research

    Article Title: ErbB2 Trafficking and Degradation Associated with K48 and K63 Polyubiquitination

    doi: 10.1158/0008-5472.CAN-09-3768

    Figure Lengend Snippet: ErbB2 receptor overexpression in SK-BR-3 is regulated by both polyubiquitinating and deubiquitinating mechanisms. A. Immunofluorescence images of ErbB2 expression in SK-BR-3 cells transiently transfected with constructs overexpressing either wildtype

    Article Snippet: The mouse IgG1 monoclonal anti-c-ErbB2/c-Neu (Ab-3), developed against the C-terminal 1242–1255 amino acids of human ErbB2, was purchased from Calbiochem (San Diego, CA).

    Techniques: Over Expression, Immunofluorescence, Expressing, Transfection, Construct

    SK-BR-3 editing of ErbB2 polyubiquitin K48 and K63 linkages following proteasome or HSP90 inhibition, as monitored by MRM/MS. A. Full-length (185 kDa) ErbB2 immunoprecipitates from untreated and proteasome inhibited (PS341, 20nM) SK-BR-3 cells were resolved

    Journal: Cancer research

    Article Title: ErbB2 Trafficking and Degradation Associated with K48 and K63 Polyubiquitination

    doi: 10.1158/0008-5472.CAN-09-3768

    Figure Lengend Snippet: SK-BR-3 editing of ErbB2 polyubiquitin K48 and K63 linkages following proteasome or HSP90 inhibition, as monitored by MRM/MS. A. Full-length (185 kDa) ErbB2 immunoprecipitates from untreated and proteasome inhibited (PS341, 20nM) SK-BR-3 cells were resolved

    Article Snippet: The mouse IgG1 monoclonal anti-c-ErbB2/c-Neu (Ab-3), developed against the C-terminal 1242–1255 amino acids of human ErbB2, was purchased from Calbiochem (San Diego, CA).

    Techniques: Inhibition, Mass Spectrometry

    4T1.2neu cells have an elevated level of HER-2/ neu receptor density compared with 4T1.2 breast cancer cells. (A) In-cell Western blot shows fluorescence signal intensity after HER-2/ neu staining (top panel). Cells were normalized using DRAQ5 nuclear staining

    Journal: Translational Oncology

    Article Title: Detection of Cancer Metastases with a Dual-labeled Near-Infrared/Positron Emission Tomography Imaging Agent 1Detection of Cancer Metastases with a Dual-labeled Near-Infrared/Positron Emission Tomography Imaging Agent 1 2

    doi:

    Figure Lengend Snippet: 4T1.2neu cells have an elevated level of HER-2/ neu receptor density compared with 4T1.2 breast cancer cells. (A) In-cell Western blot shows fluorescence signal intensity after HER-2/ neu staining (top panel). Cells were normalized using DRAQ5 nuclear staining

    Article Snippet: Mouse antirat HER-2/ neu primary antibody (Calbiochem, La Jolla, CA) was diluted in blocking buffer at a concentration of 5 µg/ml and added to the cells to incubate for 2.5 hours at room temperature.

    Techniques: In-Cell ELISA, Fluorescence, Staining

    Functional collaboration between PAK4 and MMP-2 in the regulation of α v β 3/EGFR-mediated migration and invasion in glioma. ( a ) 4910 and 5310 cells were treated with mock, pSV and PAK4si as described in Materials and Methods. At 24 h post-transfection cells were further treated with EGF (2 μ g/ml) and GW2974 (10 μ M) and incubated for another 24 h. Western blotting was performed using whole-cell lysates and GAPDH served as loading control. ( b ) Combination treatments were performed with VN (5 μ g/ml) and α v β 3 integrin-blocking antibody (5 μ g/ml) as described above and representative blots form three independent experiments were shown. ( c ) Percentage of cell invasion obtained from three independent experiments were plotted in the bar diagram (* P

    Journal: Cell Death & Disease

    Article Title: Functional cooperativity by direct interaction between PAK4 and MMP-2 in the regulation of anoikis resistance, migration and invasion in glioma

    doi: 10.1038/cddis.2012.182

    Figure Lengend Snippet: Functional collaboration between PAK4 and MMP-2 in the regulation of α v β 3/EGFR-mediated migration and invasion in glioma. ( a ) 4910 and 5310 cells were treated with mock, pSV and PAK4si as described in Materials and Methods. At 24 h post-transfection cells were further treated with EGF (2 μ g/ml) and GW2974 (10 μ M) and incubated for another 24 h. Western blotting was performed using whole-cell lysates and GAPDH served as loading control. ( b ) Combination treatments were performed with VN (5 μ g/ml) and α v β 3 integrin-blocking antibody (5 μ g/ml) as described above and representative blots form three independent experiments were shown. ( c ) Percentage of cell invasion obtained from three independent experiments were plotted in the bar diagram (* P

    Article Snippet: Additionally, EGFR/ErbB2 dual inhibitor-GW2974 (Sigma Aldrich, St. Louis, MO, USA), recombinant human-EGF protein and α vβ 3-integrin function-blocking antibody (clone 23C6, Millipore Inc., Billerica, MA, USA) were used in the present study.

    Techniques: Functional Assay, Migration, Transfection, Incubation, Western Blot, Blocking Assay

    Depletion of LRIG1 augments ErbB2 expression and enhances cellular proliferation. A , Lysates from MDA-MB-453 and BT474 cells transfected for 72h with RNAi oligos targeting LRIG1 (LRIG1-KD) or scrambled control (SC) were blotted with antibodies to p-ErbB2, ErbB2, LRIG1, and actin. B , 48h after transfecting with RNAi oligos, MDA-MB-453 cells were serum starved overnight and then treated with increasing concentrations of Nrg1 for 15 min. Whole cell lysates were blotted with antibodies to LRIG1, pAKT, tAKT, pERK, tERK, and tubulin as a loading control. The bottom panel plots the densitometic analysis of the average pAKT signal from three independent experiments for each Nrg1 concentration along with SE. C , 24h after transfecting cells with RNAi oligos as described above, MDA-MB-453 and BT474 cells were placed in serum starve media, in the absence or presence of 2.5 nM Nrg1, or complete media (10% FCS). After 48h, proliferation was measured by an MTT assay. Three independent experiments were conducted with a representative experiment shown. Error bars represent SE of four replicates within this experiment. D , 24h after transfecting cells with RNAi oligos as described above, MDA-MB-453 cells were placed in serum starve or complete media in the absence or presence of the EGFR/ErbB2 inhibitor 4557W. After 48h, proliferation was measured by an MTT assay, with basal proliferation being normalized as 100%. Additionally, whole cell lysates from serum starved cells were collected following treatment and blotted for p-ErbB2 and actin (top panel) to verify inhibition of ErbB2 by 4557W.

    Journal: Cancer research

    Article Title: Suppression of the negative regulator LRIG1 contributes to ErbB2 overexpression in breast cancer

    doi: 10.1158/0008-5472.CAN-07-6316

    Figure Lengend Snippet: Depletion of LRIG1 augments ErbB2 expression and enhances cellular proliferation. A , Lysates from MDA-MB-453 and BT474 cells transfected for 72h with RNAi oligos targeting LRIG1 (LRIG1-KD) or scrambled control (SC) were blotted with antibodies to p-ErbB2, ErbB2, LRIG1, and actin. B , 48h after transfecting with RNAi oligos, MDA-MB-453 cells were serum starved overnight and then treated with increasing concentrations of Nrg1 for 15 min. Whole cell lysates were blotted with antibodies to LRIG1, pAKT, tAKT, pERK, tERK, and tubulin as a loading control. The bottom panel plots the densitometic analysis of the average pAKT signal from three independent experiments for each Nrg1 concentration along with SE. C , 24h after transfecting cells with RNAi oligos as described above, MDA-MB-453 and BT474 cells were placed in serum starve media, in the absence or presence of 2.5 nM Nrg1, or complete media (10% FCS). After 48h, proliferation was measured by an MTT assay. Three independent experiments were conducted with a representative experiment shown. Error bars represent SE of four replicates within this experiment. D , 24h after transfecting cells with RNAi oligos as described above, MDA-MB-453 cells were placed in serum starve or complete media in the absence or presence of the EGFR/ErbB2 inhibitor 4557W. After 48h, proliferation was measured by an MTT assay, with basal proliferation being normalized as 100%. Additionally, whole cell lysates from serum starved cells were collected following treatment and blotted for p-ErbB2 and actin (top panel) to verify inhibition of ErbB2 by 4557W.

    Article Snippet: The EGFR/ErbB2 inhibitor 4557W was purchased from EMD Bioscience (San Diego, CA).

    Techniques: Expressing, Multiple Displacement Amplification, Transfection, Concentration Assay, MTT Assay, Inhibition