rabbit polyclonal anti phospho erbb 2  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc rabbit polyclonal anti phospho erbb 2
    ( A, B ) MCF-7 cells were transfected with control siRNA or siRNAs directed against IKKα, IKKβ or IKKγ. 48 hours after transfection, cells were incubated in the absence (−) or presence (+) of 150 nM Sema4D (A) or Sema4C (B) for 20 minutes, lysed, and the amount of activated RhoA and R-Ras as well as their respective expression levels were determined as described in Materials and Methods , or cells were lysed and <t>ErbB-2</t> phosphorylation was visualised using a specific anti-phospho-ErbB-2 antibody. Equal protein expression levels in cell lysates were confirmed by immunoblotting using an anti-ErbB-2 antibody. ( C ) MCF-7 cells incubated in the absence (−) or presence (+) of 25 nM Sema4D were simultaneously stimulated with increasing concentrations of TNFα for 20 minutes, lysed and ErbB-2 phosphorylation was visualized using a specific anti-phospho-ErbB-2 antibody. Shown are representative examples of at least three experiments. ( D ) MCF-7 reporter cells were treated without (−) or with (+) 25 ng/ml of TNFα. Simultaneously, cells were incubated with increasing concentrations of Sema4D (as indicated) for 8 hours, and SRE luciferase activity was quantified. Shown are the mean values of three indpendent experiments −/+ SD. *, P<0.05.
    Rabbit Polyclonal Anti Phospho Erbb 2, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 97/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/rabbit polyclonal anti phospho erbb 2/product/Cell Signaling Technology Inc
    Average 97 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    rabbit polyclonal anti phospho erbb 2 - by Bioz Stars, 2023-03
    97/100 stars

    Images

    1) Product Images from "The IκB Kinase Complex Is Required for Plexin-B-Mediated Activation of RhoA"

    Article Title: The IκB Kinase Complex Is Required for Plexin-B-Mediated Activation of RhoA

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0105661

    ( A, B ) MCF-7 cells were transfected with control siRNA or siRNAs directed against IKKα, IKKβ or IKKγ. 48 hours after transfection, cells were incubated in the absence (−) or presence (+) of 150 nM Sema4D (A) or Sema4C (B) for 20 minutes, lysed, and the amount of activated RhoA and R-Ras as well as their respective expression levels were determined as described in Materials and Methods , or cells were lysed and ErbB-2 phosphorylation was visualised using a specific anti-phospho-ErbB-2 antibody. Equal protein expression levels in cell lysates were confirmed by immunoblotting using an anti-ErbB-2 antibody. ( C ) MCF-7 cells incubated in the absence (−) or presence (+) of 25 nM Sema4D were simultaneously stimulated with increasing concentrations of TNFα for 20 minutes, lysed and ErbB-2 phosphorylation was visualized using a specific anti-phospho-ErbB-2 antibody. Shown are representative examples of at least three experiments. ( D ) MCF-7 reporter cells were treated without (−) or with (+) 25 ng/ml of TNFα. Simultaneously, cells were incubated with increasing concentrations of Sema4D (as indicated) for 8 hours, and SRE luciferase activity was quantified. Shown are the mean values of three indpendent experiments −/+ SD. *, P<0.05.
    Figure Legend Snippet: ( A, B ) MCF-7 cells were transfected with control siRNA or siRNAs directed against IKKα, IKKβ or IKKγ. 48 hours after transfection, cells were incubated in the absence (−) or presence (+) of 150 nM Sema4D (A) or Sema4C (B) for 20 minutes, lysed, and the amount of activated RhoA and R-Ras as well as their respective expression levels were determined as described in Materials and Methods , or cells were lysed and ErbB-2 phosphorylation was visualised using a specific anti-phospho-ErbB-2 antibody. Equal protein expression levels in cell lysates were confirmed by immunoblotting using an anti-ErbB-2 antibody. ( C ) MCF-7 cells incubated in the absence (−) or presence (+) of 25 nM Sema4D were simultaneously stimulated with increasing concentrations of TNFα for 20 minutes, lysed and ErbB-2 phosphorylation was visualized using a specific anti-phospho-ErbB-2 antibody. Shown are representative examples of at least three experiments. ( D ) MCF-7 reporter cells were treated without (−) or with (+) 25 ng/ml of TNFα. Simultaneously, cells were incubated with increasing concentrations of Sema4D (as indicated) for 8 hours, and SRE luciferase activity was quantified. Shown are the mean values of three indpendent experiments −/+ SD. *, P<0.05.

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

    ( A, B ) HEK-293 cells were transfected with cDNAs encoding VSV-Plexin-B1 (VSV-PlxnB1), FLAG-PDZ-RhoGEF (PRG) alone or together with kinase-deficient mutants of HA-tagged IKKα (K44M) (IKKα-KD) or HA-tagged IKKβ (K44M) (IKKβ-KD) including SRE.L reporter (RepLucdelCMV) (A) or NF-kB-dependent luciferase reporter plasmid (NF-κB-Luc) (B). 48 hours after transfection, cells were incubated with 25 ng/ml TNFα or 150 nM Sema4D for 8 hours (as indicated), and luciferase activity was determined. Shown are the mean values of three independent experiments −/+ SD. *, P<0.05. ( C, D ) MCF-7 cells were treated with SC-514 (50 µM) or NBDBP (100 µM) for the indicated time periods. After incubation in the absence (−) or presence (+) of 150 nM Sema4D for 20 minutes (C) or 10 ng/ml EGF for 20 minutes (D), cells were lysed, and a specific antibody directed against the phosphorylated version of ErbB-2 was used to visualize ErbB-2 phosphorylation. ErbB-2 levels in lysed samples were controlled using an anti-ErbB-2 antibody. Shown are representative examples of at least three experiments.
    Figure Legend Snippet: ( A, B ) HEK-293 cells were transfected with cDNAs encoding VSV-Plexin-B1 (VSV-PlxnB1), FLAG-PDZ-RhoGEF (PRG) alone or together with kinase-deficient mutants of HA-tagged IKKα (K44M) (IKKα-KD) or HA-tagged IKKβ (K44M) (IKKβ-KD) including SRE.L reporter (RepLucdelCMV) (A) or NF-kB-dependent luciferase reporter plasmid (NF-κB-Luc) (B). 48 hours after transfection, cells were incubated with 25 ng/ml TNFα or 150 nM Sema4D for 8 hours (as indicated), and luciferase activity was determined. Shown are the mean values of three independent experiments −/+ SD. *, P<0.05. ( C, D ) MCF-7 cells were treated with SC-514 (50 µM) or NBDBP (100 µM) for the indicated time periods. After incubation in the absence (−) or presence (+) of 150 nM Sema4D for 20 minutes (C) or 10 ng/ml EGF for 20 minutes (D), cells were lysed, and a specific antibody directed against the phosphorylated version of ErbB-2 was used to visualize ErbB-2 phosphorylation. ErbB-2 levels in lysed samples were controlled using an anti-ErbB-2 antibody. Shown are representative examples of at least three experiments.

    Techniques Used: Transfection, Luciferase, Plasmid Preparation, Incubation, Activity Assay

    ( A ) After incubation with Sema4D (150 nM) or TNFα (25 ng/ml) for the indicated time periods, MCF-7 cells were lysed, and IκBα degradation was visualized using an anti-IκBα antibody. ( B ) MCF-7 cells were treated with TNFα (25 ng/ml), Sema4D (150 nM) or control buffer (PBS) for 20 minutes and lysed. IKKα/β proteins were precipitated using an anti-IKKα/β antibody. Precipitates were further processed and subjected to an in vitro kinase assay as described in Materials and Methods . A recombinant active IKKβ isoform served as positive control. Shown are the mean values of absorption measured at a wavelength of 450 nm of three independent experiments −/+ SD. *, P<0.05. ( C ) HEK-293 cells were transfected with cDNAs coding for VSV-tagged Plexin-B1 (VSV-PlxnB1), FLAG-tagged PDZ-RhoGEF (FLAG-PRG) and NF-κB-dependent luciferase reporter plasmid (NF-κB-Luc). 48 hours after transfection, cells were incubated without (−) or with (+) TNFα (25 ng/ml) or Sema4D (150 nM) for 8 hours followed by the photometric quantification of reporter luciferase activity. ( D ) Wild-type MCF-7 cells (WT) and MCF-7 cells transduced with a degradation-resistent dominant-negative IκBα mutant (S32A/S36A) were serum-depleted, incubated in the absence (−) or presence (+) of 25 ng/ml TNFα or 150 nM Sema4D for 20 minutes and lysed. Lysates were probed with anti-IκBα antibody (left panel) to test the expression and functionality of the IκBα mutant or were immunoblotted with an anti-phospho-ErbB-2 antibody to visualize phosphorylated ErbB-2 and with an anti-ErbB-2 antibody to control expression levels (right panel). Protein levels were controlled by immunoblotting with an anti-α-tubulin antibody. ( E ) MCF-7 cells were preincubated with 25 µM of NF-κB inhibitor SN50 for the indicated time periods. Thereafter, cells were treated with control buffer (−) or 150 nM Sema4D (+) for 20 minutes, lysed and ErbB-2 phosphorylation was analyzed as described (left panel). To test the functionality of the NF-κB inhibitor, HEK-293 cells were transfected with a NF-κB dependent luciferase reporter plasmid (NF-κB-Luc) (right panel). After preincubation with 25 µM SN50 for 120 minutes, HEK-293 cells were incubated in the absence (−) or presence (+) of 25 ng/ml TNFα for 8 hours and luciferase acitivity was quantified. Shown are the mean values of three independent experiments −/+ SD. *, P<0.05.
    Figure Legend Snippet: ( A ) After incubation with Sema4D (150 nM) or TNFα (25 ng/ml) for the indicated time periods, MCF-7 cells were lysed, and IκBα degradation was visualized using an anti-IκBα antibody. ( B ) MCF-7 cells were treated with TNFα (25 ng/ml), Sema4D (150 nM) or control buffer (PBS) for 20 minutes and lysed. IKKα/β proteins were precipitated using an anti-IKKα/β antibody. Precipitates were further processed and subjected to an in vitro kinase assay as described in Materials and Methods . A recombinant active IKKβ isoform served as positive control. Shown are the mean values of absorption measured at a wavelength of 450 nm of three independent experiments −/+ SD. *, P<0.05. ( C ) HEK-293 cells were transfected with cDNAs coding for VSV-tagged Plexin-B1 (VSV-PlxnB1), FLAG-tagged PDZ-RhoGEF (FLAG-PRG) and NF-κB-dependent luciferase reporter plasmid (NF-κB-Luc). 48 hours after transfection, cells were incubated without (−) or with (+) TNFα (25 ng/ml) or Sema4D (150 nM) for 8 hours followed by the photometric quantification of reporter luciferase activity. ( D ) Wild-type MCF-7 cells (WT) and MCF-7 cells transduced with a degradation-resistent dominant-negative IκBα mutant (S32A/S36A) were serum-depleted, incubated in the absence (−) or presence (+) of 25 ng/ml TNFα or 150 nM Sema4D for 20 minutes and lysed. Lysates were probed with anti-IκBα antibody (left panel) to test the expression and functionality of the IκBα mutant or were immunoblotted with an anti-phospho-ErbB-2 antibody to visualize phosphorylated ErbB-2 and with an anti-ErbB-2 antibody to control expression levels (right panel). Protein levels were controlled by immunoblotting with an anti-α-tubulin antibody. ( E ) MCF-7 cells were preincubated with 25 µM of NF-κB inhibitor SN50 for the indicated time periods. Thereafter, cells were treated with control buffer (−) or 150 nM Sema4D (+) for 20 minutes, lysed and ErbB-2 phosphorylation was analyzed as described (left panel). To test the functionality of the NF-κB inhibitor, HEK-293 cells were transfected with a NF-κB dependent luciferase reporter plasmid (NF-κB-Luc) (right panel). After preincubation with 25 µM SN50 for 120 minutes, HEK-293 cells were incubated in the absence (−) or presence (+) of 25 ng/ml TNFα for 8 hours and luciferase acitivity was quantified. Shown are the mean values of three independent experiments −/+ SD. *, P<0.05.

    Techniques Used: Incubation, In Vitro, Kinase Assay, Recombinant, Positive Control, Transfection, Luciferase, Plasmid Preparation, Activity Assay, Transduction, Dominant Negative Mutation, Mutagenesis, Expressing, Western Blot

    ( A ) HEK-293 cells were transfected with cDNAs encoding VSV-Plexin-B1 (VSV-PlxnB1), FLAG-PDZ-RhoGEF (FLAG-PRG) alone or together with a HA-tagged kinase-deficient IKKα-mutant (HA-IKKα-KD). After incubation in the absence (−) or presence (+) of 150 nM Sema4D for 20 minutes, VSV-Plexin-B1 was immunoprecipitated (IP) using an anti-VSV antibody and precipitates were immunoblotted (IB) using anti-ErbB-2, anti-VSV or anti-HA antibodies. Shown are the autoluminograms of immunoblots stained with the indicated antibodies. ( B ) MCF-7 cells were incubated with buffer (−) or IKK inhibitor SC-514 (50 µM) for 30 minutes. Thereafter, cells were stimulated without (−) or with 150 nM Sema4D (+) for 20 minutes, lysed, and endogenous Plexin-B1 was immunoprecipitated using an anti-Plexin-B1 antibody. Shown are Western blots of lysed or immunoprecipitated (IP) samples stained with the indicated antibodies (IB). ( C ) 48 hours after transfection with cDNAs encoding truncated versions of VSV-Plexin-B1 (VSV-PlxnB1ΔIC) and HA-ErbB-2 (HA-ErbB-2ΔIC), HEK293 cells were treated without (−) or with IKK inhibitor – SC-514 (50 µM) or NBDBP (100 µM) for 30 minutes. Thereafter, cells were incubated in the absence (−) or presence (+) of 150 nM Sema4D for 20 minutes, lysed and immunoprecipitated (IP) using an anti-VSV antibody. Precipitates were seperated by SDS-PAGE and analyzed by immunoblotting (IB) with anti-VSV- or anti-HA- antibodies. ( D ) MDA-MB-468 cells were incubated without (−) or with SC-514 (50 µM) or NBDBP (100 µM) for 30 minutes, and Plexin-B1 was then immunoprecipitated (IP) from lysed cells. Precipitates were analysed by SDS-PAGE and immunoblotted with antibodies against c-Met or Plexin-B1. Shown are representative examples of at least three experiments.
    Figure Legend Snippet: ( A ) HEK-293 cells were transfected with cDNAs encoding VSV-Plexin-B1 (VSV-PlxnB1), FLAG-PDZ-RhoGEF (FLAG-PRG) alone or together with a HA-tagged kinase-deficient IKKα-mutant (HA-IKKα-KD). After incubation in the absence (−) or presence (+) of 150 nM Sema4D for 20 minutes, VSV-Plexin-B1 was immunoprecipitated (IP) using an anti-VSV antibody and precipitates were immunoblotted (IB) using anti-ErbB-2, anti-VSV or anti-HA antibodies. Shown are the autoluminograms of immunoblots stained with the indicated antibodies. ( B ) MCF-7 cells were incubated with buffer (−) or IKK inhibitor SC-514 (50 µM) for 30 minutes. Thereafter, cells were stimulated without (−) or with 150 nM Sema4D (+) for 20 minutes, lysed, and endogenous Plexin-B1 was immunoprecipitated using an anti-Plexin-B1 antibody. Shown are Western blots of lysed or immunoprecipitated (IP) samples stained with the indicated antibodies (IB). ( C ) 48 hours after transfection with cDNAs encoding truncated versions of VSV-Plexin-B1 (VSV-PlxnB1ΔIC) and HA-ErbB-2 (HA-ErbB-2ΔIC), HEK293 cells were treated without (−) or with IKK inhibitor – SC-514 (50 µM) or NBDBP (100 µM) for 30 minutes. Thereafter, cells were incubated in the absence (−) or presence (+) of 150 nM Sema4D for 20 minutes, lysed and immunoprecipitated (IP) using an anti-VSV antibody. Precipitates were seperated by SDS-PAGE and analyzed by immunoblotting (IB) with anti-VSV- or anti-HA- antibodies. ( D ) MDA-MB-468 cells were incubated without (−) or with SC-514 (50 µM) or NBDBP (100 µM) for 30 minutes, and Plexin-B1 was then immunoprecipitated (IP) from lysed cells. Precipitates were analysed by SDS-PAGE and immunoblotted with antibodies against c-Met or Plexin-B1. Shown are representative examples of at least three experiments.

    Techniques Used: Transfection, Mutagenesis, Incubation, Immunoprecipitation, Western Blot, Staining, SDS Page

    rabbit polyclonal anti phospho erbb 2  (Cell Signaling Technology Inc)


    Bioz Verified Symbol Cell Signaling Technology Inc is a verified supplier
    Bioz Manufacturer Symbol Cell Signaling Technology Inc manufactures this product  
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    • 97
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    Structured Review

    Cell Signaling Technology Inc rabbit polyclonal anti phospho erbb 2
    ( A, B ) MCF-7 cells were transfected with control siRNA or siRNAs directed against IKKα, IKKβ or IKKγ. 48 hours after transfection, cells were incubated in the absence (−) or presence (+) of 150 nM Sema4D (A) or Sema4C (B) for 20 minutes, lysed, and the amount of activated RhoA and R-Ras as well as their respective expression levels were determined as described in Materials and Methods , or cells were lysed and <t>ErbB-2</t> phosphorylation was visualised using a specific anti-phospho-ErbB-2 antibody. Equal protein expression levels in cell lysates were confirmed by immunoblotting using an anti-ErbB-2 antibody. ( C ) MCF-7 cells incubated in the absence (−) or presence (+) of 25 nM Sema4D were simultaneously stimulated with increasing concentrations of TNFα for 20 minutes, lysed and ErbB-2 phosphorylation was visualized using a specific anti-phospho-ErbB-2 antibody. Shown are representative examples of at least three experiments. ( D ) MCF-7 reporter cells were treated without (−) or with (+) 25 ng/ml of TNFα. Simultaneously, cells were incubated with increasing concentrations of Sema4D (as indicated) for 8 hours, and SRE luciferase activity was quantified. Shown are the mean values of three indpendent experiments −/+ SD. *, P<0.05.
    Rabbit Polyclonal Anti Phospho Erbb 2, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 97/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/rabbit polyclonal anti phospho erbb 2/product/Cell Signaling Technology Inc
    Average 97 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    rabbit polyclonal anti phospho erbb 2 - by Bioz Stars, 2023-03
    97/100 stars

    Images

    1) Product Images from "The IκB Kinase Complex Is Required for Plexin-B-Mediated Activation of RhoA"

    Article Title: The IκB Kinase Complex Is Required for Plexin-B-Mediated Activation of RhoA

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0105661

    ( A, B ) MCF-7 cells were transfected with control siRNA or siRNAs directed against IKKα, IKKβ or IKKγ. 48 hours after transfection, cells were incubated in the absence (−) or presence (+) of 150 nM Sema4D (A) or Sema4C (B) for 20 minutes, lysed, and the amount of activated RhoA and R-Ras as well as their respective expression levels were determined as described in Materials and Methods , or cells were lysed and ErbB-2 phosphorylation was visualised using a specific anti-phospho-ErbB-2 antibody. Equal protein expression levels in cell lysates were confirmed by immunoblotting using an anti-ErbB-2 antibody. ( C ) MCF-7 cells incubated in the absence (−) or presence (+) of 25 nM Sema4D were simultaneously stimulated with increasing concentrations of TNFα for 20 minutes, lysed and ErbB-2 phosphorylation was visualized using a specific anti-phospho-ErbB-2 antibody. Shown are representative examples of at least three experiments. ( D ) MCF-7 reporter cells were treated without (−) or with (+) 25 ng/ml of TNFα. Simultaneously, cells were incubated with increasing concentrations of Sema4D (as indicated) for 8 hours, and SRE luciferase activity was quantified. Shown are the mean values of three indpendent experiments −/+ SD. *, P<0.05.
    Figure Legend Snippet: ( A, B ) MCF-7 cells were transfected with control siRNA or siRNAs directed against IKKα, IKKβ or IKKγ. 48 hours after transfection, cells were incubated in the absence (−) or presence (+) of 150 nM Sema4D (A) or Sema4C (B) for 20 minutes, lysed, and the amount of activated RhoA and R-Ras as well as their respective expression levels were determined as described in Materials and Methods , or cells were lysed and ErbB-2 phosphorylation was visualised using a specific anti-phospho-ErbB-2 antibody. Equal protein expression levels in cell lysates were confirmed by immunoblotting using an anti-ErbB-2 antibody. ( C ) MCF-7 cells incubated in the absence (−) or presence (+) of 25 nM Sema4D were simultaneously stimulated with increasing concentrations of TNFα for 20 minutes, lysed and ErbB-2 phosphorylation was visualized using a specific anti-phospho-ErbB-2 antibody. Shown are representative examples of at least three experiments. ( D ) MCF-7 reporter cells were treated without (−) or with (+) 25 ng/ml of TNFα. Simultaneously, cells were incubated with increasing concentrations of Sema4D (as indicated) for 8 hours, and SRE luciferase activity was quantified. Shown are the mean values of three indpendent experiments −/+ SD. *, P<0.05.

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

    ( A, B ) HEK-293 cells were transfected with cDNAs encoding VSV-Plexin-B1 (VSV-PlxnB1), FLAG-PDZ-RhoGEF (PRG) alone or together with kinase-deficient mutants of HA-tagged IKKα (K44M) (IKKα-KD) or HA-tagged IKKβ (K44M) (IKKβ-KD) including SRE.L reporter (RepLucdelCMV) (A) or NF-kB-dependent luciferase reporter plasmid (NF-κB-Luc) (B). 48 hours after transfection, cells were incubated with 25 ng/ml TNFα or 150 nM Sema4D for 8 hours (as indicated), and luciferase activity was determined. Shown are the mean values of three independent experiments −/+ SD. *, P<0.05. ( C, D ) MCF-7 cells were treated with SC-514 (50 µM) or NBDBP (100 µM) for the indicated time periods. After incubation in the absence (−) or presence (+) of 150 nM Sema4D for 20 minutes (C) or 10 ng/ml EGF for 20 minutes (D), cells were lysed, and a specific antibody directed against the phosphorylated version of ErbB-2 was used to visualize ErbB-2 phosphorylation. ErbB-2 levels in lysed samples were controlled using an anti-ErbB-2 antibody. Shown are representative examples of at least three experiments.
    Figure Legend Snippet: ( A, B ) HEK-293 cells were transfected with cDNAs encoding VSV-Plexin-B1 (VSV-PlxnB1), FLAG-PDZ-RhoGEF (PRG) alone or together with kinase-deficient mutants of HA-tagged IKKα (K44M) (IKKα-KD) or HA-tagged IKKβ (K44M) (IKKβ-KD) including SRE.L reporter (RepLucdelCMV) (A) or NF-kB-dependent luciferase reporter plasmid (NF-κB-Luc) (B). 48 hours after transfection, cells were incubated with 25 ng/ml TNFα or 150 nM Sema4D for 8 hours (as indicated), and luciferase activity was determined. Shown are the mean values of three independent experiments −/+ SD. *, P<0.05. ( C, D ) MCF-7 cells were treated with SC-514 (50 µM) or NBDBP (100 µM) for the indicated time periods. After incubation in the absence (−) or presence (+) of 150 nM Sema4D for 20 minutes (C) or 10 ng/ml EGF for 20 minutes (D), cells were lysed, and a specific antibody directed against the phosphorylated version of ErbB-2 was used to visualize ErbB-2 phosphorylation. ErbB-2 levels in lysed samples were controlled using an anti-ErbB-2 antibody. Shown are representative examples of at least three experiments.

    Techniques Used: Transfection, Luciferase, Plasmid Preparation, Incubation, Activity Assay

    ( A ) After incubation with Sema4D (150 nM) or TNFα (25 ng/ml) for the indicated time periods, MCF-7 cells were lysed, and IκBα degradation was visualized using an anti-IκBα antibody. ( B ) MCF-7 cells were treated with TNFα (25 ng/ml), Sema4D (150 nM) or control buffer (PBS) for 20 minutes and lysed. IKKα/β proteins were precipitated using an anti-IKKα/β antibody. Precipitates were further processed and subjected to an in vitro kinase assay as described in Materials and Methods . A recombinant active IKKβ isoform served as positive control. Shown are the mean values of absorption measured at a wavelength of 450 nm of three independent experiments −/+ SD. *, P<0.05. ( C ) HEK-293 cells were transfected with cDNAs coding for VSV-tagged Plexin-B1 (VSV-PlxnB1), FLAG-tagged PDZ-RhoGEF (FLAG-PRG) and NF-κB-dependent luciferase reporter plasmid (NF-κB-Luc). 48 hours after transfection, cells were incubated without (−) or with (+) TNFα (25 ng/ml) or Sema4D (150 nM) for 8 hours followed by the photometric quantification of reporter luciferase activity. ( D ) Wild-type MCF-7 cells (WT) and MCF-7 cells transduced with a degradation-resistent dominant-negative IκBα mutant (S32A/S36A) were serum-depleted, incubated in the absence (−) or presence (+) of 25 ng/ml TNFα or 150 nM Sema4D for 20 minutes and lysed. Lysates were probed with anti-IκBα antibody (left panel) to test the expression and functionality of the IκBα mutant or were immunoblotted with an anti-phospho-ErbB-2 antibody to visualize phosphorylated ErbB-2 and with an anti-ErbB-2 antibody to control expression levels (right panel). Protein levels were controlled by immunoblotting with an anti-α-tubulin antibody. ( E ) MCF-7 cells were preincubated with 25 µM of NF-κB inhibitor SN50 for the indicated time periods. Thereafter, cells were treated with control buffer (−) or 150 nM Sema4D (+) for 20 minutes, lysed and ErbB-2 phosphorylation was analyzed as described (left panel). To test the functionality of the NF-κB inhibitor, HEK-293 cells were transfected with a NF-κB dependent luciferase reporter plasmid (NF-κB-Luc) (right panel). After preincubation with 25 µM SN50 for 120 minutes, HEK-293 cells were incubated in the absence (−) or presence (+) of 25 ng/ml TNFα for 8 hours and luciferase acitivity was quantified. Shown are the mean values of three independent experiments −/+ SD. *, P<0.05.
    Figure Legend Snippet: ( A ) After incubation with Sema4D (150 nM) or TNFα (25 ng/ml) for the indicated time periods, MCF-7 cells were lysed, and IκBα degradation was visualized using an anti-IκBα antibody. ( B ) MCF-7 cells were treated with TNFα (25 ng/ml), Sema4D (150 nM) or control buffer (PBS) for 20 minutes and lysed. IKKα/β proteins were precipitated using an anti-IKKα/β antibody. Precipitates were further processed and subjected to an in vitro kinase assay as described in Materials and Methods . A recombinant active IKKβ isoform served as positive control. Shown are the mean values of absorption measured at a wavelength of 450 nm of three independent experiments −/+ SD. *, P<0.05. ( C ) HEK-293 cells were transfected with cDNAs coding for VSV-tagged Plexin-B1 (VSV-PlxnB1), FLAG-tagged PDZ-RhoGEF (FLAG-PRG) and NF-κB-dependent luciferase reporter plasmid (NF-κB-Luc). 48 hours after transfection, cells were incubated without (−) or with (+) TNFα (25 ng/ml) or Sema4D (150 nM) for 8 hours followed by the photometric quantification of reporter luciferase activity. ( D ) Wild-type MCF-7 cells (WT) and MCF-7 cells transduced with a degradation-resistent dominant-negative IκBα mutant (S32A/S36A) were serum-depleted, incubated in the absence (−) or presence (+) of 25 ng/ml TNFα or 150 nM Sema4D for 20 minutes and lysed. Lysates were probed with anti-IκBα antibody (left panel) to test the expression and functionality of the IκBα mutant or were immunoblotted with an anti-phospho-ErbB-2 antibody to visualize phosphorylated ErbB-2 and with an anti-ErbB-2 antibody to control expression levels (right panel). Protein levels were controlled by immunoblotting with an anti-α-tubulin antibody. ( E ) MCF-7 cells were preincubated with 25 µM of NF-κB inhibitor SN50 for the indicated time periods. Thereafter, cells were treated with control buffer (−) or 150 nM Sema4D (+) for 20 minutes, lysed and ErbB-2 phosphorylation was analyzed as described (left panel). To test the functionality of the NF-κB inhibitor, HEK-293 cells were transfected with a NF-κB dependent luciferase reporter plasmid (NF-κB-Luc) (right panel). After preincubation with 25 µM SN50 for 120 minutes, HEK-293 cells were incubated in the absence (−) or presence (+) of 25 ng/ml TNFα for 8 hours and luciferase acitivity was quantified. Shown are the mean values of three independent experiments −/+ SD. *, P<0.05.

    Techniques Used: Incubation, In Vitro, Kinase Assay, Recombinant, Positive Control, Transfection, Luciferase, Plasmid Preparation, Activity Assay, Transduction, Dominant Negative Mutation, Mutagenesis, Expressing, Western Blot

    ( A ) HEK-293 cells were transfected with cDNAs encoding VSV-Plexin-B1 (VSV-PlxnB1), FLAG-PDZ-RhoGEF (FLAG-PRG) alone or together with a HA-tagged kinase-deficient IKKα-mutant (HA-IKKα-KD). After incubation in the absence (−) or presence (+) of 150 nM Sema4D for 20 minutes, VSV-Plexin-B1 was immunoprecipitated (IP) using an anti-VSV antibody and precipitates were immunoblotted (IB) using anti-ErbB-2, anti-VSV or anti-HA antibodies. Shown are the autoluminograms of immunoblots stained with the indicated antibodies. ( B ) MCF-7 cells were incubated with buffer (−) or IKK inhibitor SC-514 (50 µM) for 30 minutes. Thereafter, cells were stimulated without (−) or with 150 nM Sema4D (+) for 20 minutes, lysed, and endogenous Plexin-B1 was immunoprecipitated using an anti-Plexin-B1 antibody. Shown are Western blots of lysed or immunoprecipitated (IP) samples stained with the indicated antibodies (IB). ( C ) 48 hours after transfection with cDNAs encoding truncated versions of VSV-Plexin-B1 (VSV-PlxnB1ΔIC) and HA-ErbB-2 (HA-ErbB-2ΔIC), HEK293 cells were treated without (−) or with IKK inhibitor – SC-514 (50 µM) or NBDBP (100 µM) for 30 minutes. Thereafter, cells were incubated in the absence (−) or presence (+) of 150 nM Sema4D for 20 minutes, lysed and immunoprecipitated (IP) using an anti-VSV antibody. Precipitates were seperated by SDS-PAGE and analyzed by immunoblotting (IB) with anti-VSV- or anti-HA- antibodies. ( D ) MDA-MB-468 cells were incubated without (−) or with SC-514 (50 µM) or NBDBP (100 µM) for 30 minutes, and Plexin-B1 was then immunoprecipitated (IP) from lysed cells. Precipitates were analysed by SDS-PAGE and immunoblotted with antibodies against c-Met or Plexin-B1. Shown are representative examples of at least three experiments.
    Figure Legend Snippet: ( A ) HEK-293 cells were transfected with cDNAs encoding VSV-Plexin-B1 (VSV-PlxnB1), FLAG-PDZ-RhoGEF (FLAG-PRG) alone or together with a HA-tagged kinase-deficient IKKα-mutant (HA-IKKα-KD). After incubation in the absence (−) or presence (+) of 150 nM Sema4D for 20 minutes, VSV-Plexin-B1 was immunoprecipitated (IP) using an anti-VSV antibody and precipitates were immunoblotted (IB) using anti-ErbB-2, anti-VSV or anti-HA antibodies. Shown are the autoluminograms of immunoblots stained with the indicated antibodies. ( B ) MCF-7 cells were incubated with buffer (−) or IKK inhibitor SC-514 (50 µM) for 30 minutes. Thereafter, cells were stimulated without (−) or with 150 nM Sema4D (+) for 20 minutes, lysed, and endogenous Plexin-B1 was immunoprecipitated using an anti-Plexin-B1 antibody. Shown are Western blots of lysed or immunoprecipitated (IP) samples stained with the indicated antibodies (IB). ( C ) 48 hours after transfection with cDNAs encoding truncated versions of VSV-Plexin-B1 (VSV-PlxnB1ΔIC) and HA-ErbB-2 (HA-ErbB-2ΔIC), HEK293 cells were treated without (−) or with IKK inhibitor – SC-514 (50 µM) or NBDBP (100 µM) for 30 minutes. Thereafter, cells were incubated in the absence (−) or presence (+) of 150 nM Sema4D for 20 minutes, lysed and immunoprecipitated (IP) using an anti-VSV antibody. Precipitates were seperated by SDS-PAGE and analyzed by immunoblotting (IB) with anti-VSV- or anti-HA- antibodies. ( D ) MDA-MB-468 cells were incubated without (−) or with SC-514 (50 µM) or NBDBP (100 µM) for 30 minutes, and Plexin-B1 was then immunoprecipitated (IP) from lysed cells. Precipitates were analysed by SDS-PAGE and immunoblotted with antibodies against c-Met or Plexin-B1. Shown are representative examples of at least three experiments.

    Techniques Used: Transfection, Mutagenesis, Incubation, Immunoprecipitation, Western Blot, Staining, SDS Page

    phospho her2 tyr1248 rabbit polyclonal antibody  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc phospho her2 tyr1248 rabbit polyclonal antibody
    SHP-1 binds to EGFR and HER-2 protein. Notes: ( A , B ) SKBR3 cells were transfected with control (NC), SHP-1 WT, or SHP-1 MT vector for 48 hours. EGFR and HER-2 proteins were pulled down by immunoprecipitation and the protein level was detected by immunoblotting. ( C , D ) SKBR3 cells were transfected with control (NC), SHP-1 WT, or SHP-1 MT vector for 48 hours. Phosphorylated-EGFR (pEGFR) and phosphorylated-HER-2 (pHER-2) proteins were pulled down by immunoprecipitation and the protein level was detected by immunoblotting. ( E , F ) The binding between SHP-1 and HER-3 or HER-4 was also evaluated through immunoprecipitation. The whole gel is shown, and no specific band of HER-3 or HER-4 was detected. ( G ) Bimolecular fluorescence complementation (BiFC) was performed to confirm the binding activity between SHP-1 and EGFR, HER-2, HER-3, and HER-4. BiFC signals were generated by interaction of the GFP fluorophore components based on proximity. + and − indicate receiving and withholding of the treatment, respectively. Abbreviations: EGFR, epidermal growth factor receptor; HER-2, human epidermal receptor; NC, nonsense control; WT, wild-type; IP, immunoprecipitation.
    Phospho Her2 Tyr1248 Rabbit Polyclonal Antibody, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Images

    1) Product Images from "Protein tyrosine phosphatase SHP-1 sensitizes EGFR/HER-2 positive breast cancer cells to trastuzumab through modulating phosphorylation of EGFR and HER-2"

    Article Title: Protein tyrosine phosphatase SHP-1 sensitizes EGFR/HER-2 positive breast cancer cells to trastuzumab through modulating phosphorylation of EGFR and HER-2

    Journal: OncoTargets and therapy

    doi: 10.2147/OTT.S82225

    SHP-1 binds to EGFR and HER-2 protein. Notes: ( A , B ) SKBR3 cells were transfected with control (NC), SHP-1 WT, or SHP-1 MT vector for 48 hours. EGFR and HER-2 proteins were pulled down by immunoprecipitation and the protein level was detected by immunoblotting. ( C , D ) SKBR3 cells were transfected with control (NC), SHP-1 WT, or SHP-1 MT vector for 48 hours. Phosphorylated-EGFR (pEGFR) and phosphorylated-HER-2 (pHER-2) proteins were pulled down by immunoprecipitation and the protein level was detected by immunoblotting. ( E , F ) The binding between SHP-1 and HER-3 or HER-4 was also evaluated through immunoprecipitation. The whole gel is shown, and no specific band of HER-3 or HER-4 was detected. ( G ) Bimolecular fluorescence complementation (BiFC) was performed to confirm the binding activity between SHP-1 and EGFR, HER-2, HER-3, and HER-4. BiFC signals were generated by interaction of the GFP fluorophore components based on proximity. + and − indicate receiving and withholding of the treatment, respectively. Abbreviations: EGFR, epidermal growth factor receptor; HER-2, human epidermal receptor; NC, nonsense control; WT, wild-type; IP, immunoprecipitation.
    Figure Legend Snippet: SHP-1 binds to EGFR and HER-2 protein. Notes: ( A , B ) SKBR3 cells were transfected with control (NC), SHP-1 WT, or SHP-1 MT vector for 48 hours. EGFR and HER-2 proteins were pulled down by immunoprecipitation and the protein level was detected by immunoblotting. ( C , D ) SKBR3 cells were transfected with control (NC), SHP-1 WT, or SHP-1 MT vector for 48 hours. Phosphorylated-EGFR (pEGFR) and phosphorylated-HER-2 (pHER-2) proteins were pulled down by immunoprecipitation and the protein level was detected by immunoblotting. ( E , F ) The binding between SHP-1 and HER-3 or HER-4 was also evaluated through immunoprecipitation. The whole gel is shown, and no specific band of HER-3 or HER-4 was detected. ( G ) Bimolecular fluorescence complementation (BiFC) was performed to confirm the binding activity between SHP-1 and EGFR, HER-2, HER-3, and HER-4. BiFC signals were generated by interaction of the GFP fluorophore components based on proximity. + and − indicate receiving and withholding of the treatment, respectively. Abbreviations: EGFR, epidermal growth factor receptor; HER-2, human epidermal receptor; NC, nonsense control; WT, wild-type; IP, immunoprecipitation.

    Techniques Used: Transfection, Plasmid Preparation, Immunoprecipitation, Western Blot, Binding Assay, Fluorescence, Activity Assay, Generated

    rabbit polyclonal anti phospho her2 erbb2 tyr1248 antibody  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc rabbit polyclonal anti phospho her2 erbb2 tyr1248 antibody
    (A) Chromatograms of BT474 cellular metabolites extracted from control and lapatinib (1 μM) treated cells for 2 hours at 37°C. Representative of three independent experiments, all of which showed similar results. (B) Cr kinase enzyme activity assays from the fractionated lysates of BT474 cells treated with vehicle control or 1 μM lapatinib for 2 hours at 37°C. The corresponding immunoblotting (WB) results of the fractionated samples with antibodies against MtCK1, anti-phospho-tyrosine (pTyr), PDHK1 (mitochondria marker), and β-actin (cytosol marker) are also shown. (C) Top: Immunoblots of lysates from 293T cells expressing vector control (−) and <t>HER2</t> WT. Bottom: qRT-PCR results of MtCK1 mRNA in 293T cells expressing vector control (−) or HER2 WT. (D) Immunoblots of anti-Flag immunoprecipitates from cells expressing Flag-MtCK1 WT and Y153F mutant. (E) Immunoblots of cells treated with 1 μM lapatinib for 2 hours at 37°C. MtCK1 total protein levels were normalized between the control and lapatinib-treated samples to compare the levels of phospho-Y153 MtCK1 between the samples. All results are representative experiments of three independent replicates and P values were determined by a two-tailed Student’s t test (**P<0.01).
    Rabbit Polyclonal Anti Phospho Her2 Erbb2 Tyr1248 Antibody, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Images

    1) Product Images from "Tyrosine Phosphorylation of Mitochondrial Creatine Kinase 1 Enhances a Druggable Tumor Energy Shuttle Pathway"

    Article Title: Tyrosine Phosphorylation of Mitochondrial Creatine Kinase 1 Enhances a Druggable Tumor Energy Shuttle Pathway

    Journal: Cell metabolism

    doi: 10.1016/j.cmet.2018.08.008

    (A) Chromatograms of BT474 cellular metabolites extracted from control and lapatinib (1 μM) treated cells for 2 hours at 37°C. Representative of three independent experiments, all of which showed similar results. (B) Cr kinase enzyme activity assays from the fractionated lysates of BT474 cells treated with vehicle control or 1 μM lapatinib for 2 hours at 37°C. The corresponding immunoblotting (WB) results of the fractionated samples with antibodies against MtCK1, anti-phospho-tyrosine (pTyr), PDHK1 (mitochondria marker), and β-actin (cytosol marker) are also shown. (C) Top: Immunoblots of lysates from 293T cells expressing vector control (−) and HER2 WT. Bottom: qRT-PCR results of MtCK1 mRNA in 293T cells expressing vector control (−) or HER2 WT. (D) Immunoblots of anti-Flag immunoprecipitates from cells expressing Flag-MtCK1 WT and Y153F mutant. (E) Immunoblots of cells treated with 1 μM lapatinib for 2 hours at 37°C. MtCK1 total protein levels were normalized between the control and lapatinib-treated samples to compare the levels of phospho-Y153 MtCK1 between the samples. All results are representative experiments of three independent replicates and P values were determined by a two-tailed Student’s t test (**P<0.01).
    Figure Legend Snippet: (A) Chromatograms of BT474 cellular metabolites extracted from control and lapatinib (1 μM) treated cells for 2 hours at 37°C. Representative of three independent experiments, all of which showed similar results. (B) Cr kinase enzyme activity assays from the fractionated lysates of BT474 cells treated with vehicle control or 1 μM lapatinib for 2 hours at 37°C. The corresponding immunoblotting (WB) results of the fractionated samples with antibodies against MtCK1, anti-phospho-tyrosine (pTyr), PDHK1 (mitochondria marker), and β-actin (cytosol marker) are also shown. (C) Top: Immunoblots of lysates from 293T cells expressing vector control (−) and HER2 WT. Bottom: qRT-PCR results of MtCK1 mRNA in 293T cells expressing vector control (−) or HER2 WT. (D) Immunoblots of anti-Flag immunoprecipitates from cells expressing Flag-MtCK1 WT and Y153F mutant. (E) Immunoblots of cells treated with 1 μM lapatinib for 2 hours at 37°C. MtCK1 total protein levels were normalized between the control and lapatinib-treated samples to compare the levels of phospho-Y153 MtCK1 between the samples. All results are representative experiments of three independent replicates and P values were determined by a two-tailed Student’s t test (**P<0.01).

    Techniques Used: Activity Assay, Western Blot, Marker, Expressing, Plasmid Preparation, Quantitative RT-PCR, Mutagenesis, Two Tailed Test

    (A) Top: Immunoblots of lysates from 293T cells co-expressing Flag-MtCK1 WT or Y153F along with vector control (−) or HER2 WT. Bottom: Densitometric quantification of four independent anti-Flag and anti-β-actin immunoblots based on the densitometric analysis showing a dose-dependent linear correlation of anti-Flag and anti-β-actin immunoblots of the lysates (Figure S2A). (B) Top: Immunoblots of SKBR3 cells stably expressing Flag-MtCK1 WT or Y153F that were treated with 200 μg/ml cycloheximide (CHX) for the indicated times. Bottom: Densitometric quantification of three independent anti-Flag and anti-β-actin immunoblots. (C) Left: MtCK1 enzyme activity in anti-Flag immunoprecipitates from 293T cells co-expressing Flag-MtCK1 WT, MtCK1 Y153F or MtCK1 E227L and HER2. Right: Immunoblots of the corresponding anti-Flag immunoprecipitates (IP) and whole-cell lysates (WCL) from 293T cells. (D) Immunoblots of 293T cells co-expressing either vector control (−) or HER2 and Flag-MtCK1 E227L. All results are representative experiments of three independent replicates. P values were determined by a two-tailed Student’s t test (*P<0.05, **P<0.01, ***p < 0.001).
    Figure Legend Snippet: (A) Top: Immunoblots of lysates from 293T cells co-expressing Flag-MtCK1 WT or Y153F along with vector control (−) or HER2 WT. Bottom: Densitometric quantification of four independent anti-Flag and anti-β-actin immunoblots based on the densitometric analysis showing a dose-dependent linear correlation of anti-Flag and anti-β-actin immunoblots of the lysates (Figure S2A). (B) Top: Immunoblots of SKBR3 cells stably expressing Flag-MtCK1 WT or Y153F that were treated with 200 μg/ml cycloheximide (CHX) for the indicated times. Bottom: Densitometric quantification of three independent anti-Flag and anti-β-actin immunoblots. (C) Left: MtCK1 enzyme activity in anti-Flag immunoprecipitates from 293T cells co-expressing Flag-MtCK1 WT, MtCK1 Y153F or MtCK1 E227L and HER2. Right: Immunoblots of the corresponding anti-Flag immunoprecipitates (IP) and whole-cell lysates (WCL) from 293T cells. (D) Immunoblots of 293T cells co-expressing either vector control (−) or HER2 and Flag-MtCK1 E227L. All results are representative experiments of three independent replicates. P values were determined by a two-tailed Student’s t test (*P<0.05, **P<0.01, ***p < 0.001).

    Techniques Used: Western Blot, Expressing, Plasmid Preparation, Stable Transfection, Activity Assay, Two Tailed Test

    (A) Immunoblots of lysates (left) and qRT-PCR for MtCK1 mRNA (right) from “rescued” BT474 cells that stably express Flag-MtCK1 WT or Y153F with stable knockdown of endogenous MtCK1. (B) Immunoblots of “rescued” BT474 cells expressing Flag-MtCK1 WT and Y153F after treatment with MG132 for 6 h. (C) Immunoblots of anti-Flag immunoprecipitates and whole-cell lysates (WCL) from Flag-MtCK1 WT and Y153F “rescued” BT474 cells. (D) Immunoblots (WB) of anti-TRAP1 or anti-IgG immunoprecipitates and whole-cell lysates (WCL) from BT474 cells with or without lapatinib treatment (500 nM, 6 h). (E) Immunoblots (WB) of anti-Flag immunoprecipitates and WCL from 293T cells expressing Flag-MtCK1 WT expressing control vector (−) or HER2. (F) Immunoblots of lysates (left) and PCr levels (right) from 293T cells expressing vector control (−) or HER2 with or without stable knockdown of endogenous TRAP1. All results are representative experiments of three independent replicates. P values were determined by a two-tailed Student’s t test (*P<0.05).
    Figure Legend Snippet: (A) Immunoblots of lysates (left) and qRT-PCR for MtCK1 mRNA (right) from “rescued” BT474 cells that stably express Flag-MtCK1 WT or Y153F with stable knockdown of endogenous MtCK1. (B) Immunoblots of “rescued” BT474 cells expressing Flag-MtCK1 WT and Y153F after treatment with MG132 for 6 h. (C) Immunoblots of anti-Flag immunoprecipitates and whole-cell lysates (WCL) from Flag-MtCK1 WT and Y153F “rescued” BT474 cells. (D) Immunoblots (WB) of anti-TRAP1 or anti-IgG immunoprecipitates and whole-cell lysates (WCL) from BT474 cells with or without lapatinib treatment (500 nM, 6 h). (E) Immunoblots (WB) of anti-Flag immunoprecipitates and WCL from 293T cells expressing Flag-MtCK1 WT expressing control vector (−) or HER2. (F) Immunoblots of lysates (left) and PCr levels (right) from 293T cells expressing vector control (−) or HER2 with or without stable knockdown of endogenous TRAP1. All results are representative experiments of three independent replicates. P values were determined by a two-tailed Student’s t test (*P<0.05).

    Techniques Used: Western Blot, Quantitative RT-PCR, Stable Transfection, Expressing, Plasmid Preparation, Two Tailed Test

    (A) Immunoblots of an in vitro tyrosine kinase assay using recombinant ABL with purified recombinant MtCK1 WT protein. (B) Immunoblots of in vitro ABL kinase assay using recombinant ABL and MtCK1 WT or Y153F proteins. (C) Immunoblots of anti-Flag immunoprecipitates and WCL from 293T cells co-expressing Flag-MtCK1 WT and vector control (−), HER2 WT or ABL. (D) Immunoblots of BT474 cells treated with DMSO, or imatinib (20 βM), or lapatinib (500 nM) for 6 h. (E) Immunoblots of anti-Flag immunoprecipitates and WCL from BT474 cells treated with imatinib (10 βM) overnight. (F) Immunoblots of anti-TRAP1 or anti-IgG immunoprecipitates and WCL from BT474 cells with or without imatinib treatment (20 βM) for 6 h. (G) Immunoblots of anti-IgG control and anti-MtCK1 antibody immunoprecipitates and input lysate from BT474 cells. (H) Isolated mitochondria from BT474 cells were treated with 5 μg/mL proteinase K in the absence and presence of 1% Triton X-100 and immunoblotted for the indicated proteins. All immunoblots results are representative experiments of three independent replicates.
    Figure Legend Snippet: (A) Immunoblots of an in vitro tyrosine kinase assay using recombinant ABL with purified recombinant MtCK1 WT protein. (B) Immunoblots of in vitro ABL kinase assay using recombinant ABL and MtCK1 WT or Y153F proteins. (C) Immunoblots of anti-Flag immunoprecipitates and WCL from 293T cells co-expressing Flag-MtCK1 WT and vector control (−), HER2 WT or ABL. (D) Immunoblots of BT474 cells treated with DMSO, or imatinib (20 βM), or lapatinib (500 nM) for 6 h. (E) Immunoblots of anti-Flag immunoprecipitates and WCL from BT474 cells treated with imatinib (10 βM) overnight. (F) Immunoblots of anti-TRAP1 or anti-IgG immunoprecipitates and WCL from BT474 cells with or without imatinib treatment (20 βM) for 6 h. (G) Immunoblots of anti-IgG control and anti-MtCK1 antibody immunoprecipitates and input lysate from BT474 cells. (H) Isolated mitochondria from BT474 cells were treated with 5 μg/mL proteinase K in the absence and presence of 1% Triton X-100 and immunoblotted for the indicated proteins. All immunoblots results are representative experiments of three independent replicates.

    Techniques Used: Western Blot, In Vitro, Tyrosine Kinase Assay, Recombinant, Purification, Kinase Assay, Expressing, Plasmid Preparation, Isolation

    (A) Immunoblots of three normal breast tissue lysates (#1-#3), six HER2+ patient-derived xenograft (PDX) tumor lysates (#4-#9), five TNBC PDX tumor lysates (#10-#14). (B) Tumor volumes in NSG mice (N=5) with PDX #4 cells with or without MtCK1 knockdown. Each dot represents tumor volume in an individual mouse with error bars ± standard deviation. Immunoblots of lysates from the corresponding cells are also shown. (C) Structures of Cr and Cyclo-Cr. (D) HPLC analysis of metabolites extracted from SKBR3-R cells treated with either water or Cyclo-Cr (1 mM) overnight. The experiment shown is representative of three independent experiments, all of which showed similar results. (E) Clonogenic assay results of breast cancer cell lines treated with Cyclo-Cr. (F) Cell proliferation assay results of SKBR3-R treated with Cyclo-Cr and/or PCr. (G) Cell proliferation assay results of normal primary mammary epithelial cells up to 6 mM of CCr. Error bars in E-G ± standard deviation of 3 independent measurements. P values were determined by a two-tailed Student’s t test. ***P<0.001.
    Figure Legend Snippet: (A) Immunoblots of three normal breast tissue lysates (#1-#3), six HER2+ patient-derived xenograft (PDX) tumor lysates (#4-#9), five TNBC PDX tumor lysates (#10-#14). (B) Tumor volumes in NSG mice (N=5) with PDX #4 cells with or without MtCK1 knockdown. Each dot represents tumor volume in an individual mouse with error bars ± standard deviation. Immunoblots of lysates from the corresponding cells are also shown. (C) Structures of Cr and Cyclo-Cr. (D) HPLC analysis of metabolites extracted from SKBR3-R cells treated with either water or Cyclo-Cr (1 mM) overnight. The experiment shown is representative of three independent experiments, all of which showed similar results. (E) Clonogenic assay results of breast cancer cell lines treated with Cyclo-Cr. (F) Cell proliferation assay results of SKBR3-R treated with Cyclo-Cr and/or PCr. (G) Cell proliferation assay results of normal primary mammary epithelial cells up to 6 mM of CCr. Error bars in E-G ± standard deviation of 3 independent measurements. P values were determined by a two-tailed Student’s t test. ***P<0.001.

    Techniques Used: Western Blot, Derivative Assay, Standard Deviation, Clonogenic Assay, Proliferation Assay, Two Tailed Test

    (A) Tumor growth in nude mice with BT474-R tumors treated with 0.3% Cyclo-Cr in drinking water versus regular drinking water. The Cyclo-Cr and PCr concentrations in the harvested BT474-R tumors determined by HPLC analysis are also shown below. N=8 in each group. (B) Tumor growth of HER2+ PDX #4 in NSG mice treated with 0.3% Cyclo-Cr in drinking water versus regular drinking water. The Cyclo-Cr and PCr concentrations in harvested PDX #4 tumors determined by HPLC analysis are also shown below. N=6 in each group. (C) Left: Tumor growth in NSG mice with trastuzumab-resistant HER2+ PDX #4 under various drug combinations (a total of 6 groups; control, Cyclo-Cr alone, lapatinib alone, Cyclo-Cr plus PCr, Cyclo-Cr plus lapatinib, and Cyclo-Cr plus lapatinib plus PCr). Cyclo-Cr was administered in drinking water at 0.3%, lapatinib was administrated orally at 50 mg/kg/day and PCr was administered at 400 mg/kg/day intraperitoneally. Right: Bar graphs represent tumor volume in mice with various treatments at day 20. N=9 in each group. P values were determined by a two-tailed Student’s t test. *P<0.05, **P<0.01, ***P<0.001.
    Figure Legend Snippet: (A) Tumor growth in nude mice with BT474-R tumors treated with 0.3% Cyclo-Cr in drinking water versus regular drinking water. The Cyclo-Cr and PCr concentrations in the harvested BT474-R tumors determined by HPLC analysis are also shown below. N=8 in each group. (B) Tumor growth of HER2+ PDX #4 in NSG mice treated with 0.3% Cyclo-Cr in drinking water versus regular drinking water. The Cyclo-Cr and PCr concentrations in harvested PDX #4 tumors determined by HPLC analysis are also shown below. N=6 in each group. (C) Left: Tumor growth in NSG mice with trastuzumab-resistant HER2+ PDX #4 under various drug combinations (a total of 6 groups; control, Cyclo-Cr alone, lapatinib alone, Cyclo-Cr plus PCr, Cyclo-Cr plus lapatinib, and Cyclo-Cr plus lapatinib plus PCr). Cyclo-Cr was administered in drinking water at 0.3%, lapatinib was administrated orally at 50 mg/kg/day and PCr was administered at 400 mg/kg/day intraperitoneally. Right: Bar graphs represent tumor volume in mice with various treatments at day 20. N=9 in each group. P values were determined by a two-tailed Student’s t test. *P<0.05, **P<0.01, ***P<0.001.

    Techniques Used: Two Tailed Test

    KEY RESOURCES TABLE
    Figure Legend Snippet: KEY RESOURCES TABLE

    Techniques Used: Derivative Assay, Recombinant, FLAG-tag, Plasmid Preparation, shRNA, Software, Clone Assay, Mutagenesis, SYBR Green Assay, CyQUANT Assay, Proliferation Assay, Cell Viability Assay, Protease Inhibitor

    rabbit polyclonal anti phospho her2 erbb2 tyr 1248  (Cell Signaling Technology Inc)


    Bioz Verified Symbol Cell Signaling Technology Inc is a verified supplier
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    Cell Signaling Technology Inc rabbit polyclonal anti phospho her2 erbb2 tyr 1248
    (A) Sequences of the TM and JM regions of WT EGFR and <t>ErbB2.</t> GXXXG motifs are noted with orange lines. Conserved residues are highlighted in yellow. JM residues substituted with Cys in CCH-1 or CCH-10 ErbB2 are indicated with green (M663 and L667) or blue dots (R665 and Q668), respectively. (B) Representative TIRF-M images of CHO-K1 cells expressing FLAG-tagged WT or V635G CCH-1 and CCH-10 ErbB2 after ReAsH treatment. Scale bars represent 10 μm. (C and D) Quantification of TIRF-M results, from “n” number of cells, as a fold increase in expression-corrected ReAsH fluorescence over background of CHO-K1 cells expressing WT, CCH-1, or CCH-10 ErbB2 (with/without a V635G mutation in the TM domain). Error bars represent SEM. ***p<0.001 and ****p<0.0001 from one-way ANOVA with Bonferroni post-analysis accounting for multiple comparisons. See also Figure S6 and Table S1.
    Rabbit Polyclonal Anti Phospho Her2 Erbb2 Tyr 1248, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 97/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/rabbit polyclonal anti phospho her2 erbb2 tyr 1248/product/Cell Signaling Technology Inc
    Average 97 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
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    Images

    1) Product Images from "Mechanism of allosteric coupling into and through the plasma membrane by EGFR"

    Article Title: Mechanism of allosteric coupling into and through the plasma membrane by EGFR

    Journal: Cell chemical biology

    doi: 10.1016/j.chembiol.2018.04.005

    (A) Sequences of the TM and JM regions of WT EGFR and ErbB2. GXXXG motifs are noted with orange lines. Conserved residues are highlighted in yellow. JM residues substituted with Cys in CCH-1 or CCH-10 ErbB2 are indicated with green (M663 and L667) or blue dots (R665 and Q668), respectively. (B) Representative TIRF-M images of CHO-K1 cells expressing FLAG-tagged WT or V635G CCH-1 and CCH-10 ErbB2 after ReAsH treatment. Scale bars represent 10 μm. (C and D) Quantification of TIRF-M results, from “n” number of cells, as a fold increase in expression-corrected ReAsH fluorescence over background of CHO-K1 cells expressing WT, CCH-1, or CCH-10 ErbB2 (with/without a V635G mutation in the TM domain). Error bars represent SEM. ***p<0.001 and ****p<0.0001 from one-way ANOVA with Bonferroni post-analysis accounting for multiple comparisons. See also Figure S6 and Table S1.
    Figure Legend Snippet: (A) Sequences of the TM and JM regions of WT EGFR and ErbB2. GXXXG motifs are noted with orange lines. Conserved residues are highlighted in yellow. JM residues substituted with Cys in CCH-1 or CCH-10 ErbB2 are indicated with green (M663 and L667) or blue dots (R665 and Q668), respectively. (B) Representative TIRF-M images of CHO-K1 cells expressing FLAG-tagged WT or V635G CCH-1 and CCH-10 ErbB2 after ReAsH treatment. Scale bars represent 10 μm. (C and D) Quantification of TIRF-M results, from “n” number of cells, as a fold increase in expression-corrected ReAsH fluorescence over background of CHO-K1 cells expressing WT, CCH-1, or CCH-10 ErbB2 (with/without a V635G mutation in the TM domain). Error bars represent SEM. ***p<0.001 and ****p<0.0001 from one-way ANOVA with Bonferroni post-analysis accounting for multiple comparisons. See also Figure S6 and Table S1.

    Techniques Used: Expressing, Fluorescence, Mutagenesis

    KEY RESOURCES TABLE
    Figure Legend Snippet: KEY RESOURCES TABLE

    Techniques Used: Recombinant, Protease Inhibitor, Binding Assay, Western Blot, Mutagenesis, Fluorescence, Live Cell Imaging, Software

    rabbit polyclonal  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc rabbit polyclonal
    Rabbit Polyclonal, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    rabbit polyclonal anti phospho egfr py992  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc rabbit polyclonal anti phospho egfr py992

    Rabbit Polyclonal Anti Phospho Egfr Py992, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 97/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "Context Specificity in Causal Signaling Networks Revealed by Phosphoprotein Profiling"

    Article Title: Context Specificity in Causal Signaling Networks Revealed by Phosphoprotein Profiling

    Journal: Cell Systems

    doi: 10.1016/j.cels.2016.11.013


    Figure Legend Snippet:

    Techniques Used: Transduction, Recombinant, Protease Inhibitor, Bicinchoninic Acid Protein Assay, Software, Inhibition, Modification

    polyclonal rabbit anti phospho erbb2  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc polyclonal rabbit anti phospho erbb2
    A. Left panel , visualization of the interaction between <t>ErbB2</t> and nucleolin (red dots) in naïve MDCK cells and MDCK ErbB2-expressing clones was performed using a proximity ligation assay (PLA). Right panel , differences between signal intensity in both cell lines represented as the number of dots per cell (means ±SE). B. Left panel , visualization of the interaction between ErbB2 and nucleolin (red dots) in SKBR3 untreated cells and cells treated with either control siRNA or ErbB2-specific siRNA. Upper right panel , a western-blot analysis of ErbB2-siRNA treatment effect on ErbB2 levels in SKBR3 cells; numbers below bands indicate average fold induction of untreated. Lower right panel , differences in signal intensity represented as number of dots per cell (means ±SE).
    Polyclonal Rabbit Anti Phospho Erbb2, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 97/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "Nucleolin-binding by ErbB2 enhances tumorigenicity of ErbB2-positive breast cancer"

    Article Title: Nucleolin-binding by ErbB2 enhances tumorigenicity of ErbB2-positive breast cancer

    Journal: Oncotarget

    doi: 10.18632/oncotarget.11323

    A. Left panel , visualization of the interaction between ErbB2 and nucleolin (red dots) in naïve MDCK cells and MDCK ErbB2-expressing clones was performed using a proximity ligation assay (PLA). Right panel , differences between signal intensity in both cell lines represented as the number of dots per cell (means ±SE). B. Left panel , visualization of the interaction between ErbB2 and nucleolin (red dots) in SKBR3 untreated cells and cells treated with either control siRNA or ErbB2-specific siRNA. Upper right panel , a western-blot analysis of ErbB2-siRNA treatment effect on ErbB2 levels in SKBR3 cells; numbers below bands indicate average fold induction of untreated. Lower right panel , differences in signal intensity represented as number of dots per cell (means ±SE).
    Figure Legend Snippet: A. Left panel , visualization of the interaction between ErbB2 and nucleolin (red dots) in naïve MDCK cells and MDCK ErbB2-expressing clones was performed using a proximity ligation assay (PLA). Right panel , differences between signal intensity in both cell lines represented as the number of dots per cell (means ±SE). B. Left panel , visualization of the interaction between ErbB2 and nucleolin (red dots) in SKBR3 untreated cells and cells treated with either control siRNA or ErbB2-specific siRNA. Upper right panel , a western-blot analysis of ErbB2-siRNA treatment effect on ErbB2 levels in SKBR3 cells; numbers below bands indicate average fold induction of untreated. Lower right panel , differences in signal intensity represented as number of dots per cell (means ±SE).

    Techniques Used: Expressing, Clone Assay, Proximity Ligation Assay, Western Blot

    A. Colony formation was tested in either naïve MDCK cells or MDCK stable clones expressing ErbB2 and nucleolin, either separately or in combination. Upper panel , representative images. Lower panel , total colonies area presented as fold induction of control (means ±SD). B. MDCK cells overexpressing nucleolin, ErbB2, nucleolin and ErbB2 or none were plated and grown in soft agar, as described in Materials and Methods. Upper panel , photomicrographs of typical wells. Lower panel , number of colonies (size>0.01 mm 2 ; means ±SD). *** ( p-value <0.005) - ErbB2 and nucleolin clone compared to the controls; ^^^ ( p-value <0.005) - ErbB2/nucleolin clones compared to naïve control.
    Figure Legend Snippet: A. Colony formation was tested in either naïve MDCK cells or MDCK stable clones expressing ErbB2 and nucleolin, either separately or in combination. Upper panel , representative images. Lower panel , total colonies area presented as fold induction of control (means ±SD). B. MDCK cells overexpressing nucleolin, ErbB2, nucleolin and ErbB2 or none were plated and grown in soft agar, as described in Materials and Methods. Upper panel , photomicrographs of typical wells. Lower panel , number of colonies (size>0.01 mm 2 ; means ±SD). *** ( p-value <0.005) - ErbB2 and nucleolin clone compared to the controls; ^^^ ( p-value <0.005) - ErbB2/nucleolin clones compared to naïve control.

    Techniques Used: Clone Assay, Expressing

    A. Schematic representation of full-length (wt) nucleolin and its truncation variants: N-ter, 212, RBD and GAR. B. Co-immunoprecipitation (co-IP) analysis of ErbB2 and full-length nucleolin (NCL), N-ter and 212. HEK-293T cells were co-transfected with ErbB2 and either nucleolin variants or control vectors. C. Co-IP analysis of ErbB2 and the C-terminal variants of nucleolin: 212, RBD and GAR. HEK-293T cells were co-transfected with ErbB2 and either 212 (as a positive control), RBD, GAR or control vectors.
    Figure Legend Snippet: A. Schematic representation of full-length (wt) nucleolin and its truncation variants: N-ter, 212, RBD and GAR. B. Co-immunoprecipitation (co-IP) analysis of ErbB2 and full-length nucleolin (NCL), N-ter and 212. HEK-293T cells were co-transfected with ErbB2 and either nucleolin variants or control vectors. C. Co-IP analysis of ErbB2 and the C-terminal variants of nucleolin: 212, RBD and GAR. HEK-293T cells were co-transfected with ErbB2 and either 212 (as a positive control), RBD, GAR or control vectors.

    Techniques Used: Immunoprecipitation, Co-Immunoprecipitation Assay, Transfection, Positive Control

    A. Schematic representation of full-length (wt) ErbB2 and its mutants: Δcyt-NLS and ΔNLS. B. Co-IP analysis of nucleolin and ErbB2 mutants. HEK-293T cells were co-transfected with Flag-tagged nucleolin and either wt ErbB2, ΔNLS, Δcyt-NLS or control vectors.
    Figure Legend Snippet: A. Schematic representation of full-length (wt) ErbB2 and its mutants: Δcyt-NLS and ΔNLS. B. Co-IP analysis of nucleolin and ErbB2 mutants. HEK-293T cells were co-transfected with Flag-tagged nucleolin and either wt ErbB2, ΔNLS, Δcyt-NLS or control vectors.

    Techniques Used: Co-Immunoprecipitation Assay, Transfection

    Phosphorylation levels of ErbB2 were determined by western-blotting using an anti-phospho-ErbB2 antibody. A. SKBR3 cancer cells were transiently transfected with anti-nucleolin shRNA; numbers below bands indicate average fold induction of nucleolin relative to untreated cells (means ±SD). B. HEK-293T cells were transiently transfected with ErbB2 and either wt nucleolin, N-ter, 212 or a control vector (means ±SD). C. HEK-293T cells were transiently transfected with ErbB2 or ΔNLS and either wt nucleolin or a control vector (means ±SD; *** ( p-value <0.005) - nucleolin-transfected compared to GFP-transfected control; ^^ ( p-value <0.01) -ΔNLS-transfected compared to wt ErbB2-transfected cells). D. HEK-293T cells were transiently transfected with either ErbB1 alone or in combination with wt ErbB2 or ΔNLS, and total phosphorylation levels of Y20 were measured by western-blotting, prior to and following EGF-stimulation (10min), as indicated (means ±SD; *** ( p-value <0.005) - EGF-stimulated compared to unstimulated cells; ^^^ ( p-value <0.005) - comparison between co-transfections in cells either unstimulated or stimulated with EGF).
    Figure Legend Snippet: Phosphorylation levels of ErbB2 were determined by western-blotting using an anti-phospho-ErbB2 antibody. A. SKBR3 cancer cells were transiently transfected with anti-nucleolin shRNA; numbers below bands indicate average fold induction of nucleolin relative to untreated cells (means ±SD). B. HEK-293T cells were transiently transfected with ErbB2 and either wt nucleolin, N-ter, 212 or a control vector (means ±SD). C. HEK-293T cells were transiently transfected with ErbB2 or ΔNLS and either wt nucleolin or a control vector (means ±SD; *** ( p-value <0.005) - nucleolin-transfected compared to GFP-transfected control; ^^ ( p-value <0.01) -ΔNLS-transfected compared to wt ErbB2-transfected cells). D. HEK-293T cells were transiently transfected with either ErbB1 alone or in combination with wt ErbB2 or ΔNLS, and total phosphorylation levels of Y20 were measured by western-blotting, prior to and following EGF-stimulation (10min), as indicated (means ±SD; *** ( p-value <0.005) - EGF-stimulated compared to unstimulated cells; ^^^ ( p-value <0.005) - comparison between co-transfections in cells either unstimulated or stimulated with EGF).

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

    A. Western-blot analysis of activation levels of ErbB2 downstream signaling pathways, as determined by phosphorylation of Erk and Akt proteins in HEK-293T cells transiently transfected with ErbB2, nucleolin, neither or both (means ±SD). B. Activation of ErbB2 by nucleolin and its downstream signaling was inhibited by transient overexpression of GFP-TM-NLS peptides in stably GFP or GFP-nucleolin-overexpressing SKBR3 breast cancer cells (means ±SD). C. SKBR3 cells were transiently transfected with either GFP-TM-NLS or a control, and cell viability was measured using the methylene blue assay (means ±SD).
    Figure Legend Snippet: A. Western-blot analysis of activation levels of ErbB2 downstream signaling pathways, as determined by phosphorylation of Erk and Akt proteins in HEK-293T cells transiently transfected with ErbB2, nucleolin, neither or both (means ±SD). B. Activation of ErbB2 by nucleolin and its downstream signaling was inhibited by transient overexpression of GFP-TM-NLS peptides in stably GFP or GFP-nucleolin-overexpressing SKBR3 breast cancer cells (means ±SD). C. SKBR3 cells were transiently transfected with either GFP-TM-NLS or a control, and cell viability was measured using the methylene blue assay (means ±SD).

    Techniques Used: Western Blot, Activation Assay, Transfection, Over Expression, Stable Transfection

    A. Kaplan-Meier curves depicting survival rates of patients overexpressing either ErbB2 alone or both ErbB2 and nucleolin (n=64). B. Cox-proportional hazards model for ErbB2-positive breast cancer patients, with or without nucleolin overexpression (n=305). C. Age of disease onset in patients overexpressing either ErbB2 alone or both ErbB2 and nucleolin (n=518). D. Time from initial diagnosis until detection of an additional, new, tumor (metastatic or primary) in the same patient (n=42). Data was compared between patients overexpressing ErbB2 and patients overexpressing ErbB2 and nucleolin.
    Figure Legend Snippet: A. Kaplan-Meier curves depicting survival rates of patients overexpressing either ErbB2 alone or both ErbB2 and nucleolin (n=64). B. Cox-proportional hazards model for ErbB2-positive breast cancer patients, with or without nucleolin overexpression (n=305). C. Age of disease onset in patients overexpressing either ErbB2 alone or both ErbB2 and nucleolin (n=518). D. Time from initial diagnosis until detection of an additional, new, tumor (metastatic or primary) in the same patient (n=42). Data was compared between patients overexpressing ErbB2 and patients overexpressing ErbB2 and nucleolin.

    Techniques Used: Over Expression

    A. SKBR3 cells were treated with GroA (10μM), and cell viability was measured by the methylene blue assay at the indicated time points (means ±SD). B. Western-blot analysis of ErbB2 activation, determined by ErbB2 phosphorylation levels, following treatment with GroA (means ±SD). C. SKBR3 cells overexpressing either GFP-nucleolin (SKBR-NCL) or GFP (SKBR-GFP) were treated with GroA (10μM), and cell viability was measured using the methylene blue assay. Left panel , time course curves of cell viability following treatment in SKBR-GFP (control) and SKBR-NCL cells. Right panel , comparison of cell viability in SKBR-GFP and SKBR-NCL cells after 5 days of treatment. Results presented as means ±SD. D. The IC-10, 20 and 50 values for GroA treatment were determined for SKBR-NCL and SKBR-GFP cells using the methylene blue viability assay (means ±SD). E. Cells were pre-treated with GroA as indicated, and total area of colonies formed was determined (means ±SD; *** ( p-value <0.005) - SKBR-GFP compared to SKBR-NCL cells; ^^^ ( p-value <0.005) - comparison between untreated and treated cells of the same cell line).
    Figure Legend Snippet: A. SKBR3 cells were treated with GroA (10μM), and cell viability was measured by the methylene blue assay at the indicated time points (means ±SD). B. Western-blot analysis of ErbB2 activation, determined by ErbB2 phosphorylation levels, following treatment with GroA (means ±SD). C. SKBR3 cells overexpressing either GFP-nucleolin (SKBR-NCL) or GFP (SKBR-GFP) were treated with GroA (10μM), and cell viability was measured using the methylene blue assay. Left panel , time course curves of cell viability following treatment in SKBR-GFP (control) and SKBR-NCL cells. Right panel , comparison of cell viability in SKBR-GFP and SKBR-NCL cells after 5 days of treatment. Results presented as means ±SD. D. The IC-10, 20 and 50 values for GroA treatment were determined for SKBR-NCL and SKBR-GFP cells using the methylene blue viability assay (means ±SD). E. Cells were pre-treated with GroA as indicated, and total area of colonies formed was determined (means ±SD; *** ( p-value <0.005) - SKBR-GFP compared to SKBR-NCL cells; ^^^ ( p-value <0.005) - comparison between untreated and treated cells of the same cell line).

    Techniques Used: Western Blot, Activation Assay, Viability Assay

    anti phosphorylationspecific polyclonal rabbit  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc anti phosphorylationspecific polyclonal rabbit
    Anti Phosphorylationspecific Polyclonal Rabbit, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    rabbit polyclonal antiphospho egfr y1148 antibodies  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc rabbit polyclonal antiphospho egfr y1148 antibodies
    Rabbit Polyclonal Antiphospho Egfr Y1148 Antibodies, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 97/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    rabbit polyclonal anti phospho erbb 2  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc rabbit polyclonal anti phospho erbb 2
    Migratory and invasive capabilities of different LNCaP cells and the activated levels of <t>ErbB-2</t> and matriptase in those cells. A: Migratory and invasive capabilities of C-33 and C-81 LNCaP cells. After trypsinization, 4 × 105 of the cells were seeded in the upper chamber of each transwell coated without or with matrigel (30 μg/cm2) in serum-free RPMI 1640 medium, and the lower chambers were filled with 5% FBS RPMI medium. Transwell migration assay was carried out for 24 hours. Migratory and invasive cells were fixed in methanol and stained with 0.25% crystal violet. Each assay was performed in triplicate for calculation of means ± SE. B: Analysis of ErbB-2 expression and phosphorylation levels by immunoblotting assays. Cell lysates were collected with 0.5% NP-40 in HEPES buffer. The ErbB-2 tyrosine phosphorylation levels were detected by using anti-phospho-ErbB-2 Tyr877 and anti-phospho-ErbB-2 Tyr-1221/Tyr-1222 antibodies (Abs). Total ErbB-2 protein level was determined by using an anti-ErbB-2 Ab (C18). C: Whole cell lysates were immunoprecipitated with an anti-ErbB-2 antibody followed by immunoblotting with anti-pTyr (4G10) and anti-ErbB-2 Abs. D: Analysis of the levels of total and activated matriptase and HAI-1 in different LNCaP cells by immunoblotting assay. LNCaP cells were lysed in 1% Triton X-100 PBS and then collected under nonboiling and nonreducing conditions. The immunoblots for total matriptase, activated matriptase, and HAI-1 were conducted by using anti-total matriptase (M32), anti-activated matriptase (M69), and anti-HAI-1 (M19) mAbs.
    Rabbit Polyclonal Anti Phospho Erbb 2, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 97/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "Matriptase Is Involved in ErbB-2-Induced Prostate Cancer Cell Invasion"

    Article Title: Matriptase Is Involved in ErbB-2-Induced Prostate Cancer Cell Invasion

    Journal:

    doi: 10.2353/ajpath.2010.100228

    Migratory and invasive capabilities of different LNCaP cells and the activated levels of ErbB-2 and matriptase in those cells. A: Migratory and invasive capabilities of C-33 and C-81 LNCaP cells. After trypsinization, 4 × 105 of the cells were seeded in the upper chamber of each transwell coated without or with matrigel (30 μg/cm2) in serum-free RPMI 1640 medium, and the lower chambers were filled with 5% FBS RPMI medium. Transwell migration assay was carried out for 24 hours. Migratory and invasive cells were fixed in methanol and stained with 0.25% crystal violet. Each assay was performed in triplicate for calculation of means ± SE. B: Analysis of ErbB-2 expression and phosphorylation levels by immunoblotting assays. Cell lysates were collected with 0.5% NP-40 in HEPES buffer. The ErbB-2 tyrosine phosphorylation levels were detected by using anti-phospho-ErbB-2 Tyr877 and anti-phospho-ErbB-2 Tyr-1221/Tyr-1222 antibodies (Abs). Total ErbB-2 protein level was determined by using an anti-ErbB-2 Ab (C18). C: Whole cell lysates were immunoprecipitated with an anti-ErbB-2 antibody followed by immunoblotting with anti-pTyr (4G10) and anti-ErbB-2 Abs. D: Analysis of the levels of total and activated matriptase and HAI-1 in different LNCaP cells by immunoblotting assay. LNCaP cells were lysed in 1% Triton X-100 PBS and then collected under nonboiling and nonreducing conditions. The immunoblots for total matriptase, activated matriptase, and HAI-1 were conducted by using anti-total matriptase (M32), anti-activated matriptase (M69), and anti-HAI-1 (M19) mAbs.
    Figure Legend Snippet: Migratory and invasive capabilities of different LNCaP cells and the activated levels of ErbB-2 and matriptase in those cells. A: Migratory and invasive capabilities of C-33 and C-81 LNCaP cells. After trypsinization, 4 × 105 of the cells were seeded in the upper chamber of each transwell coated without or with matrigel (30 μg/cm2) in serum-free RPMI 1640 medium, and the lower chambers were filled with 5% FBS RPMI medium. Transwell migration assay was carried out for 24 hours. Migratory and invasive cells were fixed in methanol and stained with 0.25% crystal violet. Each assay was performed in triplicate for calculation of means ± SE. B: Analysis of ErbB-2 expression and phosphorylation levels by immunoblotting assays. Cell lysates were collected with 0.5% NP-40 in HEPES buffer. The ErbB-2 tyrosine phosphorylation levels were detected by using anti-phospho-ErbB-2 Tyr877 and anti-phospho-ErbB-2 Tyr-1221/Tyr-1222 antibodies (Abs). Total ErbB-2 protein level was determined by using an anti-ErbB-2 Ab (C18). C: Whole cell lysates were immunoprecipitated with an anti-ErbB-2 antibody followed by immunoblotting with anti-pTyr (4G10) and anti-ErbB-2 Abs. D: Analysis of the levels of total and activated matriptase and HAI-1 in different LNCaP cells by immunoblotting assay. LNCaP cells were lysed in 1% Triton X-100 PBS and then collected under nonboiling and nonreducing conditions. The immunoblots for total matriptase, activated matriptase, and HAI-1 were conducted by using anti-total matriptase (M32), anti-activated matriptase (M69), and anti-HAI-1 (M19) mAbs.

    Techniques Used: Transwell Migration Assay, Staining, Expressing, Western Blot, Immunoprecipitation

    Effects of ErbB-2 level and activity on matriptase zymogen activation in PCa cells. A: Effects of ErbB-2 overexpression on matriptase activation in C-33 LNCaP cells. LNCaP C-33 cells were transiently transfected with ErbB-2 cDNA by Lipofectamine 2000 reagents, and control cells were transiently transfected with vector alone. Two days after transfection, cells were harvested for Western blot analysis. The separated proteins were transferred to a NC membrane and detected by immunoblotting with anti-pTyr (PY100), anti-ErbB-2 (C18), anti-c-Myc, and anti-β-actin (AC-15) Abs. For analyses of matriptase and HAI-1, nonreduced and nonboiled cell lysates were collected with Triton 1% X-100 in PBS, and immunoblotted with anti-total matriptase (M32) and anti-activated matriptase (M69) mAbs. B: Effects of ErbB-2 overexpression on matriptase activation in DU 145 cells. DU 145 cells were transiently transfected with ErbB-2 cDNA, and the immunoblots were performed as described in A. C: Effects of ErbB-2 knockdown on activation of matriptase in C-81 LNCaP cells. Cells were seeded at a density of 1.2 × 106 per well in 6-cm dishes. One day after plating, cells were infected with lentiviral particles containing ErbB-2 shRNA for 24 hours. Control cells were infected with lentiviral particles containing luciferase shRNA. Three days after selection with 1 μg/ml puromycin, cells were harvested for Western blot analysis. For analyses of the p-Tyr and protein levels of ErbB-2, cell lysates were collected and detected by anti-pTyr (PY100) and anti-ErbB-2 (C18) Abs. For analyses of matriptase and HAI-1, nonreduced and nonboiled cell lysates were collected with Triton 1% X-100 in PBS. Matriptase and HAI-1 were detected by immunoblotting with anti-total matriptase (M32) and anti-activated matriptase (M69) mAbs. Equal protein loading was verified by blotting the membranes with an anti-β-actin Ab (AC-15). D: Effects of ErbB-2 re-expression on matriptase activation in ErbB-2-knockdown cells. C-81 LNCaP cells were infected with viral particles with control luciferase shRNA or ErbB-2u shRNA for 3 days. The ErbB-2u shRNA was designed to target a specific sequence located in the 3′ UTR of ErbB-2. The ErbB-2- knockdown cells were transfected with control vector or ErbB-2 cDNA and cultured for 3 days. Cell lysates were collected and assayed by using an anti-ErbB-2 Ab (C18). For analyses of matriptase, cell lysates were collected as described in Figure 1D to detect the levels of total and activated matriptase by immunoblotting with anti-total matriptase (M32) and anti-activated matriptase (M69) mAbs. Equal protein loading was verified by blotting the membranes with an anti-β-actin Ab (AC-15).
    Figure Legend Snippet: Effects of ErbB-2 level and activity on matriptase zymogen activation in PCa cells. A: Effects of ErbB-2 overexpression on matriptase activation in C-33 LNCaP cells. LNCaP C-33 cells were transiently transfected with ErbB-2 cDNA by Lipofectamine 2000 reagents, and control cells were transiently transfected with vector alone. Two days after transfection, cells were harvested for Western blot analysis. The separated proteins were transferred to a NC membrane and detected by immunoblotting with anti-pTyr (PY100), anti-ErbB-2 (C18), anti-c-Myc, and anti-β-actin (AC-15) Abs. For analyses of matriptase and HAI-1, nonreduced and nonboiled cell lysates were collected with Triton 1% X-100 in PBS, and immunoblotted with anti-total matriptase (M32) and anti-activated matriptase (M69) mAbs. B: Effects of ErbB-2 overexpression on matriptase activation in DU 145 cells. DU 145 cells were transiently transfected with ErbB-2 cDNA, and the immunoblots were performed as described in A. C: Effects of ErbB-2 knockdown on activation of matriptase in C-81 LNCaP cells. Cells were seeded at a density of 1.2 × 106 per well in 6-cm dishes. One day after plating, cells were infected with lentiviral particles containing ErbB-2 shRNA for 24 hours. Control cells were infected with lentiviral particles containing luciferase shRNA. Three days after selection with 1 μg/ml puromycin, cells were harvested for Western blot analysis. For analyses of the p-Tyr and protein levels of ErbB-2, cell lysates were collected and detected by anti-pTyr (PY100) and anti-ErbB-2 (C18) Abs. For analyses of matriptase and HAI-1, nonreduced and nonboiled cell lysates were collected with Triton 1% X-100 in PBS. Matriptase and HAI-1 were detected by immunoblotting with anti-total matriptase (M32) and anti-activated matriptase (M69) mAbs. Equal protein loading was verified by blotting the membranes with an anti-β-actin Ab (AC-15). D: Effects of ErbB-2 re-expression on matriptase activation in ErbB-2-knockdown cells. C-81 LNCaP cells were infected with viral particles with control luciferase shRNA or ErbB-2u shRNA for 3 days. The ErbB-2u shRNA was designed to target a specific sequence located in the 3′ UTR of ErbB-2. The ErbB-2- knockdown cells were transfected with control vector or ErbB-2 cDNA and cultured for 3 days. Cell lysates were collected and assayed by using an anti-ErbB-2 Ab (C18). For analyses of matriptase, cell lysates were collected as described in Figure 1D to detect the levels of total and activated matriptase by immunoblotting with anti-total matriptase (M32) and anti-activated matriptase (M69) mAbs. Equal protein loading was verified by blotting the membranes with an anti-β-actin Ab (AC-15).

    Techniques Used: Activity Assay, Activation Assay, Over Expression, Transfection, Plasmid Preparation, Western Blot, Infection, shRNA, Luciferase, Selection, Expressing, Sequencing, Cell Culture

    Role of MEK/ERKs in ErbB-2-induced matriptase activation in C-33 LNCaP cells. A: Stable ErbB-2-overexpressing C-33 LNCaP cells were seeded at a density of 6 × 105 per well in a 6-well plate. Two days after plating, cells were treated with PD98059 at concentrations of 0, 10, 50, and 100 μmol/L; control transfected cells were treated with concentrations of 0 and 100 μmol/L PD98059. Treatment was carried out for 24 hours, and then cells were harvested for Western blot analysis. Cell lysates were collected with 0.5% NP-40 in HEPES buffer. The p-Tyr and protein levels of ErbB-2 were analyzed by immunoblotting with anti-pTyr (PY100) and anti-ErbB-2 (C18) Abs. The phosphorylation and protein levels of Erk1/2 were detected by anti-phosphoErk (Thr202 and Tyr204 of Erk1; Thr185 and Tyr187 of Erk2) and anti-Erk1/2 Abs. Nonreduced and nonboiled cell lysates were used for immunoblots to detect the levels of total and activated matriptase with anti-total matriptase (M32) and anti-activated matriptase (M69) mAbs. Equal loading was evaluated with an anti-β-actin Ab. B: Effects of a constitutively active MEK on matriptase activation in C-33 LNCaP cells. Cells were seeded at a density of 1.2 × 106 per well in 6-cm dishes. Two days after plating, cells were transiently transfected with a constitutively active MEK (CA-MEK) cDNA and harvested 48 hours after transfection; control cells were transfected with vector alone. Cell lysates were collected with 0.5% NP-40 in HEPES buffer. The phosphorylation and protein levels of Erk1/2 were analyzed by immunoblotting with an anti-phosphoErk (Thr202 and Tyr204 of Erk1; Thr185 and Tyr187 of Erk2) and anti-Erk1/2 Abs. Nonreduced and nonboiled cell lysates were used to assay the activation status and total matriptase levels with anti-total matriptase (M32) and anti-activated matriptase (M69) mAbs. An anti-β-actin (AC-15) Ab was used to evaluate protein loading.
    Figure Legend Snippet: Role of MEK/ERKs in ErbB-2-induced matriptase activation in C-33 LNCaP cells. A: Stable ErbB-2-overexpressing C-33 LNCaP cells were seeded at a density of 6 × 105 per well in a 6-well plate. Two days after plating, cells were treated with PD98059 at concentrations of 0, 10, 50, and 100 μmol/L; control transfected cells were treated with concentrations of 0 and 100 μmol/L PD98059. Treatment was carried out for 24 hours, and then cells were harvested for Western blot analysis. Cell lysates were collected with 0.5% NP-40 in HEPES buffer. The p-Tyr and protein levels of ErbB-2 were analyzed by immunoblotting with anti-pTyr (PY100) and anti-ErbB-2 (C18) Abs. The phosphorylation and protein levels of Erk1/2 were detected by anti-phosphoErk (Thr202 and Tyr204 of Erk1; Thr185 and Tyr187 of Erk2) and anti-Erk1/2 Abs. Nonreduced and nonboiled cell lysates were used for immunoblots to detect the levels of total and activated matriptase with anti-total matriptase (M32) and anti-activated matriptase (M69) mAbs. Equal loading was evaluated with an anti-β-actin Ab. B: Effects of a constitutively active MEK on matriptase activation in C-33 LNCaP cells. Cells were seeded at a density of 1.2 × 106 per well in 6-cm dishes. Two days after plating, cells were transiently transfected with a constitutively active MEK (CA-MEK) cDNA and harvested 48 hours after transfection; control cells were transfected with vector alone. Cell lysates were collected with 0.5% NP-40 in HEPES buffer. The phosphorylation and protein levels of Erk1/2 were analyzed by immunoblotting with an anti-phosphoErk (Thr202 and Tyr204 of Erk1; Thr185 and Tyr187 of Erk2) and anti-Erk1/2 Abs. Nonreduced and nonboiled cell lysates were used to assay the activation status and total matriptase levels with anti-total matriptase (M32) and anti-activated matriptase (M69) mAbs. An anti-β-actin (AC-15) Ab was used to evaluate protein loading.

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

    Role of PI 3 kinases/Akt in ErbB-2-induced matriptase activation in C-33 LNCaP cells. Stable ErbB-2-overexpressing C-33 LNCaP cells were seeded at a density of 6 × 105 per well in a 6-well plate. Two days after plating, cells were treated with LY294002 at concentrations of 0, 2, 10, and 20 μmol/L; control cells were treated with concentrations of 0 and 20 μmol/L PD98059. Treatment was carried out for 24 hours, and then cells were harvested for Western blot analysis. A: Cell lysates were collected with 0.5% NP-40 in HEPES buffer and evaluated by immunoblots for p-Tyr and protein levels of ErbB-2, performed as described previously. The phosphorylation and protein levels of Akt were detected by anti-phosphoAkt (Ser473) and anti-Akt1/2 Abs. B: Nonreduced and nonboiled cell lysates were used for immunoblotting to detect the activation status and total matriptase with anti-total matriptase (M32) and anti-activated matriptase (M69) mAbs. An anti-β-actin (AC-15) Ab was used to evaluate protein loading. C: Role of a constitutively activated Akt (Myr-Akt) in matriptase zymogen activation in C-33 LNCaP cells and DU 145 cells. LNCaP C-33 cells and DU 145 cells were transiently transfected with Myr-Akt and control plasmids by using Lipofectamine 2000. Two days after transfection, cell lysates were collected and analyzed by immunoblotting with anti-phosphoAkt (Ser473), anti-Akt1/2, anti-total matriptase (M32), and anti-activated matriptase (M69) Abs, respectively. D: Effect of Akt1 or Akt2 knockdown on activation of matriptase in LNCaP C-81 cells. Cells were seeded at a density of 1.2 × 106 per well in 6-cm dishes. One day after plating, cells were transfect with Akt1 shRNAs (shAkt1-1 and shAkt1-2), Akt2 shRNAs (shAkt2-1 and shAkt2-2), and control luciferase shRNA with Lipofectamine 2000. Cell lysates were collected for analysis of the levels of Akt1 and Akt2 protein by immunoblot with anti-Akt1 and anti-Akt2 Abs. For analyses of matriptase, cell lysates were collected and immunoblotted with anti-total matriptase (M32) and anti-activated matriptase (M69) mAbs. Equal protein loading was assessed with an anti-β-actin Ab (AC-15).
    Figure Legend Snippet: Role of PI 3 kinases/Akt in ErbB-2-induced matriptase activation in C-33 LNCaP cells. Stable ErbB-2-overexpressing C-33 LNCaP cells were seeded at a density of 6 × 105 per well in a 6-well plate. Two days after plating, cells were treated with LY294002 at concentrations of 0, 2, 10, and 20 μmol/L; control cells were treated with concentrations of 0 and 20 μmol/L PD98059. Treatment was carried out for 24 hours, and then cells were harvested for Western blot analysis. A: Cell lysates were collected with 0.5% NP-40 in HEPES buffer and evaluated by immunoblots for p-Tyr and protein levels of ErbB-2, performed as described previously. The phosphorylation and protein levels of Akt were detected by anti-phosphoAkt (Ser473) and anti-Akt1/2 Abs. B: Nonreduced and nonboiled cell lysates were used for immunoblotting to detect the activation status and total matriptase with anti-total matriptase (M32) and anti-activated matriptase (M69) mAbs. An anti-β-actin (AC-15) Ab was used to evaluate protein loading. C: Role of a constitutively activated Akt (Myr-Akt) in matriptase zymogen activation in C-33 LNCaP cells and DU 145 cells. LNCaP C-33 cells and DU 145 cells were transiently transfected with Myr-Akt and control plasmids by using Lipofectamine 2000. Two days after transfection, cell lysates were collected and analyzed by immunoblotting with anti-phosphoAkt (Ser473), anti-Akt1/2, anti-total matriptase (M32), and anti-activated matriptase (M69) Abs, respectively. D: Effect of Akt1 or Akt2 knockdown on activation of matriptase in LNCaP C-81 cells. Cells were seeded at a density of 1.2 × 106 per well in 6-cm dishes. One day after plating, cells were transfect with Akt1 shRNAs (shAkt1-1 and shAkt1-2), Akt2 shRNAs (shAkt2-1 and shAkt2-2), and control luciferase shRNA with Lipofectamine 2000. Cell lysates were collected for analysis of the levels of Akt1 and Akt2 protein by immunoblot with anti-Akt1 and anti-Akt2 Abs. For analyses of matriptase, cell lysates were collected and immunoblotted with anti-total matriptase (M32) and anti-activated matriptase (M69) mAbs. Equal protein loading was assessed with an anti-β-actin Ab (AC-15).

    Techniques Used: Activation Assay, Western Blot, Transfection, Luciferase, shRNA

    Effects of matriptase knockdown on ErbB-2-promoted cell migration of prostate cancer cells. Cells were seeded at a density of 1.2 × 106 per well in 6-cm dishes. Cells were infected by lentiviral particles with shRNAs specific to matriptase for 24 hours, selected by 1 μg/ml puromycin for 72 hours and then harvested for Western blot analysis. A: Cell lysates were collected with 0.5% NP-40 in HEPES buffer. The p-Tyr and protein levels of ErbB-2 were detected by anti-pTyr (PY100) and anti-ErbB-2 (C18) Abs. Nonreduced and nonboiled cell lysates were used for immunoblotting to detect the activation status and total matriptase with anti-total matriptase (M32) and anti-activated matriptase (M69) mAbs. Loading was analyzed with an anti-β-actin mAb (AC-15). B: Effects of matriptase knockdown on ErbB-2-promoting cell motility by wound-healing assays. Wounds with widths of approximately 250 μm were made by scraping by using 10-μl pipette tips. Cells were incubated for 24 hours for wound-healing assay. Images were captured by a light microscopy with a magnification of 100×. The dotted lines define the edges of the wounds. Migratory distances (widths at 0 hours to widths at 24 hours) were analyzed by a NIS-Elements D software (Nikon) and are represented as means ± SE calculated from triplicates; a statistically significant difference (*P < 0.05) was observed between Vec/shLuc and ErbB-2/shLuc. C: Effects of matriptase knockdown on ErbB-2-promoting cell motility by transmigration assays. After trypsinization, 1 × 105 cells were seeded with serum-free RPMI 1640 medium in each of the upper chambers, and the lower chambers were filled with 10% FBS RPMI 1640 medium. Transwell migration assay was carried out for 48 hours. Migratory cells were fixed in methanol and stained with 1% crystal violet, and images were captured by a light microscopy (original magnification, ×100). Amounts of migratory cells on each filter were counted from eight random fields (original magnification, ×200). Each assay was performed in triplicate for calculation of means ± SE; a statistically significant difference, *P < 0.05 was observed between Vec shLuc and ErbB-2 shLuc.
    Figure Legend Snippet: Effects of matriptase knockdown on ErbB-2-promoted cell migration of prostate cancer cells. Cells were seeded at a density of 1.2 × 106 per well in 6-cm dishes. Cells were infected by lentiviral particles with shRNAs specific to matriptase for 24 hours, selected by 1 μg/ml puromycin for 72 hours and then harvested for Western blot analysis. A: Cell lysates were collected with 0.5% NP-40 in HEPES buffer. The p-Tyr and protein levels of ErbB-2 were detected by anti-pTyr (PY100) and anti-ErbB-2 (C18) Abs. Nonreduced and nonboiled cell lysates were used for immunoblotting to detect the activation status and total matriptase with anti-total matriptase (M32) and anti-activated matriptase (M69) mAbs. Loading was analyzed with an anti-β-actin mAb (AC-15). B: Effects of matriptase knockdown on ErbB-2-promoting cell motility by wound-healing assays. Wounds with widths of approximately 250 μm were made by scraping by using 10-μl pipette tips. Cells were incubated for 24 hours for wound-healing assay. Images were captured by a light microscopy with a magnification of 100×. The dotted lines define the edges of the wounds. Migratory distances (widths at 0 hours to widths at 24 hours) were analyzed by a NIS-Elements D software (Nikon) and are represented as means ± SE calculated from triplicates; a statistically significant difference (*P < 0.05) was observed between Vec/shLuc and ErbB-2/shLuc. C: Effects of matriptase knockdown on ErbB-2-promoting cell motility by transmigration assays. After trypsinization, 1 × 105 cells were seeded with serum-free RPMI 1640 medium in each of the upper chambers, and the lower chambers were filled with 10% FBS RPMI 1640 medium. Transwell migration assay was carried out for 48 hours. Migratory cells were fixed in methanol and stained with 1% crystal violet, and images were captured by a light microscopy (original magnification, ×100). Amounts of migratory cells on each filter were counted from eight random fields (original magnification, ×200). Each assay was performed in triplicate for calculation of means ± SE; a statistically significant difference, *P < 0.05 was observed between Vec shLuc and ErbB-2 shLuc.

    Techniques Used: Migration, Infection, Western Blot, Activation Assay, Transferring, Incubation, Wound Healing Assay, Light Microscopy, Software, Transmigration Assay, Transwell Migration Assay, Staining

    Invasion assay of ErbB-2-overexpressing LNCaP cells with or without matriptase knockdown. Matriptase knockdown was performed as described in Figure 6A. For cell invasion assays, each filter insert was coated with 30 μg/cm2 matrigel. After trypsinization, 1 × 105 cells were seeded with serum-free RPMI 1640 medium in each insert chamber, and lower chambers were filled with 10% FBS RPMI 1640 medium. A: Transwell invasion assays were carried out for 48 hours. Invasive cells were fixed in methanol and stained with 1% crystal violet. Images were captured by a light microscopy (original magnification, ×100). B: Numbers of invasive cells on each filter were counted from eight random fields (original magnification, ×200). Each assay was performed in triplicate for calculation of means ± SE; statistically significant differences, *P < 0.05 were observed between Vec/shLuc and ErbB-2/shLuc, as well as ErbB-2/shLuc and ErbB-2/shMTX.
    Figure Legend Snippet: Invasion assay of ErbB-2-overexpressing LNCaP cells with or without matriptase knockdown. Matriptase knockdown was performed as described in Figure 6A. For cell invasion assays, each filter insert was coated with 30 μg/cm2 matrigel. After trypsinization, 1 × 105 cells were seeded with serum-free RPMI 1640 medium in each insert chamber, and lower chambers were filled with 10% FBS RPMI 1640 medium. A: Transwell invasion assays were carried out for 48 hours. Invasive cells were fixed in methanol and stained with 1% crystal violet. Images were captured by a light microscopy (original magnification, ×100). B: Numbers of invasive cells on each filter were counted from eight random fields (original magnification, ×200). Each assay was performed in triplicate for calculation of means ± SE; statistically significant differences, *P < 0.05 were observed between Vec/shLuc and ErbB-2/shLuc, as well as ErbB-2/shLuc and ErbB-2/shMTX.

    Techniques Used: Invasion Assay, Staining, Light Microscopy

    A model for the role of matriptase in ErbB-2-driven prostate cancer cell invasion. ErbB-2 hyperactivation by EGF stimulation or receptor overexpression results in the up-regulation of matriptase zymogen activation, at least in part via PI 3 kinase, leading to enhanced prostate cancer cell invasion.
    Figure Legend Snippet: A model for the role of matriptase in ErbB-2-driven prostate cancer cell invasion. ErbB-2 hyperactivation by EGF stimulation or receptor overexpression results in the up-regulation of matriptase zymogen activation, at least in part via PI 3 kinase, leading to enhanced prostate cancer cell invasion.

    Techniques Used: Over Expression, Activation Assay

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    SHP-1 binds to EGFR and HER-2 protein. Notes: ( A , B ) SKBR3 cells were transfected with control (NC), SHP-1 WT, or SHP-1 MT vector for 48 hours. EGFR and HER-2 proteins were pulled down by immunoprecipitation and the protein level was detected by immunoblotting. ( C , D ) SKBR3 cells were transfected with control (NC), SHP-1 WT, or SHP-1 MT vector for 48 hours. Phosphorylated-EGFR (pEGFR) and phosphorylated-HER-2 (pHER-2) proteins were pulled down by immunoprecipitation and the protein level was detected by immunoblotting. ( E , F ) The binding between SHP-1 and HER-3 or HER-4 was also evaluated through immunoprecipitation. The whole gel is shown, and no specific band of HER-3 or HER-4 was detected. ( G ) Bimolecular fluorescence complementation (BiFC) was performed to confirm the binding activity between SHP-1 and EGFR, HER-2, HER-3, and HER-4. BiFC signals were generated by interaction of the GFP fluorophore components based on proximity. + and − indicate receiving and withholding of the treatment, respectively. Abbreviations: EGFR, epidermal growth factor receptor; HER-2, human epidermal receptor; NC, nonsense control; WT, wild-type; IP, immunoprecipitation.
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    (A) Chromatograms of BT474 cellular metabolites extracted from control and lapatinib (1 μM) treated cells for 2 hours at 37°C. Representative of three independent experiments, all of which showed similar results. (B) Cr kinase enzyme activity assays from the fractionated lysates of BT474 cells treated with vehicle control or 1 μM lapatinib for 2 hours at 37°C. The corresponding immunoblotting (WB) results of the fractionated samples with antibodies against MtCK1, anti-phospho-tyrosine (pTyr), PDHK1 (mitochondria marker), and β-actin (cytosol marker) are also shown. (C) Top: Immunoblots of lysates from 293T cells expressing vector control (−) and <t>HER2</t> WT. Bottom: qRT-PCR results of MtCK1 mRNA in 293T cells expressing vector control (−) or HER2 WT. (D) Immunoblots of anti-Flag immunoprecipitates from cells expressing Flag-MtCK1 WT and Y153F mutant. (E) Immunoblots of cells treated with 1 μM lapatinib for 2 hours at 37°C. MtCK1 total protein levels were normalized between the control and lapatinib-treated samples to compare the levels of phospho-Y153 MtCK1 between the samples. All results are representative experiments of three independent replicates and P values were determined by a two-tailed Student’s t test (**P<0.01).
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    A. Left panel , visualization of the interaction between <t>ErbB2</t> and nucleolin (red dots) in naïve MDCK cells and MDCK ErbB2-expressing clones was performed using a proximity ligation assay (PLA). Right panel , differences between signal intensity in both cell lines represented as the number of dots per cell (means ±SE). B. Left panel , visualization of the interaction between ErbB2 and nucleolin (red dots) in SKBR3 untreated cells and cells treated with either control siRNA or ErbB2-specific siRNA. Upper right panel , a western-blot analysis of ErbB2-siRNA treatment effect on ErbB2 levels in SKBR3 cells; numbers below bands indicate average fold induction of untreated. Lower right panel , differences in signal intensity represented as number of dots per cell (means ±SE).
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    A. Left panel , visualization of the interaction between <t>ErbB2</t> and nucleolin (red dots) in naïve MDCK cells and MDCK ErbB2-expressing clones was performed using a proximity ligation assay (PLA). Right panel , differences between signal intensity in both cell lines represented as the number of dots per cell (means ±SE). B. Left panel , visualization of the interaction between ErbB2 and nucleolin (red dots) in SKBR3 untreated cells and cells treated with either control siRNA or ErbB2-specific siRNA. Upper right panel , a western-blot analysis of ErbB2-siRNA treatment effect on ErbB2 levels in SKBR3 cells; numbers below bands indicate average fold induction of untreated. Lower right panel , differences in signal intensity represented as number of dots per cell (means ±SE).
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    ( A, B ) MCF-7 cells were transfected with control siRNA or siRNAs directed against IKKα, IKKβ or IKKγ. 48 hours after transfection, cells were incubated in the absence (−) or presence (+) of 150 nM Sema4D (A) or Sema4C (B) for 20 minutes, lysed, and the amount of activated RhoA and R-Ras as well as their respective expression levels were determined as described in Materials and Methods , or cells were lysed and ErbB-2 phosphorylation was visualised using a specific anti-phospho-ErbB-2 antibody. Equal protein expression levels in cell lysates were confirmed by immunoblotting using an anti-ErbB-2 antibody. ( C ) MCF-7 cells incubated in the absence (−) or presence (+) of 25 nM Sema4D were simultaneously stimulated with increasing concentrations of TNFα for 20 minutes, lysed and ErbB-2 phosphorylation was visualized using a specific anti-phospho-ErbB-2 antibody. Shown are representative examples of at least three experiments. ( D ) MCF-7 reporter cells were treated without (−) or with (+) 25 ng/ml of TNFα. Simultaneously, cells were incubated with increasing concentrations of Sema4D (as indicated) for 8 hours, and SRE luciferase activity was quantified. Shown are the mean values of three indpendent experiments −/+ SD. *, P<0.05.

    Journal: PLoS ONE

    Article Title: The IκB Kinase Complex Is Required for Plexin-B-Mediated Activation of RhoA

    doi: 10.1371/journal.pone.0105661

    Figure Lengend Snippet: ( A, B ) MCF-7 cells were transfected with control siRNA or siRNAs directed against IKKα, IKKβ or IKKγ. 48 hours after transfection, cells were incubated in the absence (−) or presence (+) of 150 nM Sema4D (A) or Sema4C (B) for 20 minutes, lysed, and the amount of activated RhoA and R-Ras as well as their respective expression levels were determined as described in Materials and Methods , or cells were lysed and ErbB-2 phosphorylation was visualised using a specific anti-phospho-ErbB-2 antibody. Equal protein expression levels in cell lysates were confirmed by immunoblotting using an anti-ErbB-2 antibody. ( C ) MCF-7 cells incubated in the absence (−) or presence (+) of 25 nM Sema4D were simultaneously stimulated with increasing concentrations of TNFα for 20 minutes, lysed and ErbB-2 phosphorylation was visualized using a specific anti-phospho-ErbB-2 antibody. Shown are representative examples of at least three experiments. ( D ) MCF-7 reporter cells were treated without (−) or with (+) 25 ng/ml of TNFα. Simultaneously, cells were incubated with increasing concentrations of Sema4D (as indicated) for 8 hours, and SRE luciferase activity was quantified. Shown are the mean values of three indpendent experiments −/+ SD. *, P<0.05.

    Article Snippet: The following antibodies were used and obtained from commercial sources: Mouse monoclonal anti-ErbB-2 (Invitrogen, 1∶1000), rabbit polyclonal anti-phospho-ErbB-2 (Y1248, 1∶400), rabbit monoclonal anti-RhoA (1∶400), rabbit polyclonal anti-R-Ras (1:400) and rabbit monoclonal anti-IκBα (Cell Signalling Technology, 1:400), mouse monoclonal anti-HA and mouse monoclonal anti-α-tubulin (Sigma-Aldrich, 1∶1000), goat monoclonal anti-Plexin-B1 (R&D Systems, 1∶400), rabbit polyclonal anti-IKKα/β (Santa Cruz Biotechnology, 1∶500), goat polyclonal anti-VSV-G (Thermo Scientific, 1∶1000), mouse monoclonal anti-Met (Invitrogen, 1∶1000).

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

    ( A, B ) HEK-293 cells were transfected with cDNAs encoding VSV-Plexin-B1 (VSV-PlxnB1), FLAG-PDZ-RhoGEF (PRG) alone or together with kinase-deficient mutants of HA-tagged IKKα (K44M) (IKKα-KD) or HA-tagged IKKβ (K44M) (IKKβ-KD) including SRE.L reporter (RepLucdelCMV) (A) or NF-kB-dependent luciferase reporter plasmid (NF-κB-Luc) (B). 48 hours after transfection, cells were incubated with 25 ng/ml TNFα or 150 nM Sema4D for 8 hours (as indicated), and luciferase activity was determined. Shown are the mean values of three independent experiments −/+ SD. *, P<0.05. ( C, D ) MCF-7 cells were treated with SC-514 (50 µM) or NBDBP (100 µM) for the indicated time periods. After incubation in the absence (−) or presence (+) of 150 nM Sema4D for 20 minutes (C) or 10 ng/ml EGF for 20 minutes (D), cells were lysed, and a specific antibody directed against the phosphorylated version of ErbB-2 was used to visualize ErbB-2 phosphorylation. ErbB-2 levels in lysed samples were controlled using an anti-ErbB-2 antibody. Shown are representative examples of at least three experiments.

    Journal: PLoS ONE

    Article Title: The IκB Kinase Complex Is Required for Plexin-B-Mediated Activation of RhoA

    doi: 10.1371/journal.pone.0105661

    Figure Lengend Snippet: ( A, B ) HEK-293 cells were transfected with cDNAs encoding VSV-Plexin-B1 (VSV-PlxnB1), FLAG-PDZ-RhoGEF (PRG) alone or together with kinase-deficient mutants of HA-tagged IKKα (K44M) (IKKα-KD) or HA-tagged IKKβ (K44M) (IKKβ-KD) including SRE.L reporter (RepLucdelCMV) (A) or NF-kB-dependent luciferase reporter plasmid (NF-κB-Luc) (B). 48 hours after transfection, cells were incubated with 25 ng/ml TNFα or 150 nM Sema4D for 8 hours (as indicated), and luciferase activity was determined. Shown are the mean values of three independent experiments −/+ SD. *, P<0.05. ( C, D ) MCF-7 cells were treated with SC-514 (50 µM) or NBDBP (100 µM) for the indicated time periods. After incubation in the absence (−) or presence (+) of 150 nM Sema4D for 20 minutes (C) or 10 ng/ml EGF for 20 minutes (D), cells were lysed, and a specific antibody directed against the phosphorylated version of ErbB-2 was used to visualize ErbB-2 phosphorylation. ErbB-2 levels in lysed samples were controlled using an anti-ErbB-2 antibody. Shown are representative examples of at least three experiments.

    Article Snippet: The following antibodies were used and obtained from commercial sources: Mouse monoclonal anti-ErbB-2 (Invitrogen, 1∶1000), rabbit polyclonal anti-phospho-ErbB-2 (Y1248, 1∶400), rabbit monoclonal anti-RhoA (1∶400), rabbit polyclonal anti-R-Ras (1:400) and rabbit monoclonal anti-IκBα (Cell Signalling Technology, 1:400), mouse monoclonal anti-HA and mouse monoclonal anti-α-tubulin (Sigma-Aldrich, 1∶1000), goat monoclonal anti-Plexin-B1 (R&D Systems, 1∶400), rabbit polyclonal anti-IKKα/β (Santa Cruz Biotechnology, 1∶500), goat polyclonal anti-VSV-G (Thermo Scientific, 1∶1000), mouse monoclonal anti-Met (Invitrogen, 1∶1000).

    Techniques: Transfection, Luciferase, Plasmid Preparation, Incubation, Activity Assay

    ( A ) After incubation with Sema4D (150 nM) or TNFα (25 ng/ml) for the indicated time periods, MCF-7 cells were lysed, and IκBα degradation was visualized using an anti-IκBα antibody. ( B ) MCF-7 cells were treated with TNFα (25 ng/ml), Sema4D (150 nM) or control buffer (PBS) for 20 minutes and lysed. IKKα/β proteins were precipitated using an anti-IKKα/β antibody. Precipitates were further processed and subjected to an in vitro kinase assay as described in Materials and Methods . A recombinant active IKKβ isoform served as positive control. Shown are the mean values of absorption measured at a wavelength of 450 nm of three independent experiments −/+ SD. *, P<0.05. ( C ) HEK-293 cells were transfected with cDNAs coding for VSV-tagged Plexin-B1 (VSV-PlxnB1), FLAG-tagged PDZ-RhoGEF (FLAG-PRG) and NF-κB-dependent luciferase reporter plasmid (NF-κB-Luc). 48 hours after transfection, cells were incubated without (−) or with (+) TNFα (25 ng/ml) or Sema4D (150 nM) for 8 hours followed by the photometric quantification of reporter luciferase activity. ( D ) Wild-type MCF-7 cells (WT) and MCF-7 cells transduced with a degradation-resistent dominant-negative IκBα mutant (S32A/S36A) were serum-depleted, incubated in the absence (−) or presence (+) of 25 ng/ml TNFα or 150 nM Sema4D for 20 minutes and lysed. Lysates were probed with anti-IκBα antibody (left panel) to test the expression and functionality of the IκBα mutant or were immunoblotted with an anti-phospho-ErbB-2 antibody to visualize phosphorylated ErbB-2 and with an anti-ErbB-2 antibody to control expression levels (right panel). Protein levels were controlled by immunoblotting with an anti-α-tubulin antibody. ( E ) MCF-7 cells were preincubated with 25 µM of NF-κB inhibitor SN50 for the indicated time periods. Thereafter, cells were treated with control buffer (−) or 150 nM Sema4D (+) for 20 minutes, lysed and ErbB-2 phosphorylation was analyzed as described (left panel). To test the functionality of the NF-κB inhibitor, HEK-293 cells were transfected with a NF-κB dependent luciferase reporter plasmid (NF-κB-Luc) (right panel). After preincubation with 25 µM SN50 for 120 minutes, HEK-293 cells were incubated in the absence (−) or presence (+) of 25 ng/ml TNFα for 8 hours and luciferase acitivity was quantified. Shown are the mean values of three independent experiments −/+ SD. *, P<0.05.

    Journal: PLoS ONE

    Article Title: The IκB Kinase Complex Is Required for Plexin-B-Mediated Activation of RhoA

    doi: 10.1371/journal.pone.0105661

    Figure Lengend Snippet: ( A ) After incubation with Sema4D (150 nM) or TNFα (25 ng/ml) for the indicated time periods, MCF-7 cells were lysed, and IκBα degradation was visualized using an anti-IκBα antibody. ( B ) MCF-7 cells were treated with TNFα (25 ng/ml), Sema4D (150 nM) or control buffer (PBS) for 20 minutes and lysed. IKKα/β proteins were precipitated using an anti-IKKα/β antibody. Precipitates were further processed and subjected to an in vitro kinase assay as described in Materials and Methods . A recombinant active IKKβ isoform served as positive control. Shown are the mean values of absorption measured at a wavelength of 450 nm of three independent experiments −/+ SD. *, P<0.05. ( C ) HEK-293 cells were transfected with cDNAs coding for VSV-tagged Plexin-B1 (VSV-PlxnB1), FLAG-tagged PDZ-RhoGEF (FLAG-PRG) and NF-κB-dependent luciferase reporter plasmid (NF-κB-Luc). 48 hours after transfection, cells were incubated without (−) or with (+) TNFα (25 ng/ml) or Sema4D (150 nM) for 8 hours followed by the photometric quantification of reporter luciferase activity. ( D ) Wild-type MCF-7 cells (WT) and MCF-7 cells transduced with a degradation-resistent dominant-negative IκBα mutant (S32A/S36A) were serum-depleted, incubated in the absence (−) or presence (+) of 25 ng/ml TNFα or 150 nM Sema4D for 20 minutes and lysed. Lysates were probed with anti-IκBα antibody (left panel) to test the expression and functionality of the IκBα mutant or were immunoblotted with an anti-phospho-ErbB-2 antibody to visualize phosphorylated ErbB-2 and with an anti-ErbB-2 antibody to control expression levels (right panel). Protein levels were controlled by immunoblotting with an anti-α-tubulin antibody. ( E ) MCF-7 cells were preincubated with 25 µM of NF-κB inhibitor SN50 for the indicated time periods. Thereafter, cells were treated with control buffer (−) or 150 nM Sema4D (+) for 20 minutes, lysed and ErbB-2 phosphorylation was analyzed as described (left panel). To test the functionality of the NF-κB inhibitor, HEK-293 cells were transfected with a NF-κB dependent luciferase reporter plasmid (NF-κB-Luc) (right panel). After preincubation with 25 µM SN50 for 120 minutes, HEK-293 cells were incubated in the absence (−) or presence (+) of 25 ng/ml TNFα for 8 hours and luciferase acitivity was quantified. Shown are the mean values of three independent experiments −/+ SD. *, P<0.05.

    Article Snippet: The following antibodies were used and obtained from commercial sources: Mouse monoclonal anti-ErbB-2 (Invitrogen, 1∶1000), rabbit polyclonal anti-phospho-ErbB-2 (Y1248, 1∶400), rabbit monoclonal anti-RhoA (1∶400), rabbit polyclonal anti-R-Ras (1:400) and rabbit monoclonal anti-IκBα (Cell Signalling Technology, 1:400), mouse monoclonal anti-HA and mouse monoclonal anti-α-tubulin (Sigma-Aldrich, 1∶1000), goat monoclonal anti-Plexin-B1 (R&D Systems, 1∶400), rabbit polyclonal anti-IKKα/β (Santa Cruz Biotechnology, 1∶500), goat polyclonal anti-VSV-G (Thermo Scientific, 1∶1000), mouse monoclonal anti-Met (Invitrogen, 1∶1000).

    Techniques: Incubation, In Vitro, Kinase Assay, Recombinant, Positive Control, Transfection, Luciferase, Plasmid Preparation, Activity Assay, Transduction, Dominant Negative Mutation, Mutagenesis, Expressing, Western Blot

    ( A ) HEK-293 cells were transfected with cDNAs encoding VSV-Plexin-B1 (VSV-PlxnB1), FLAG-PDZ-RhoGEF (FLAG-PRG) alone or together with a HA-tagged kinase-deficient IKKα-mutant (HA-IKKα-KD). After incubation in the absence (−) or presence (+) of 150 nM Sema4D for 20 minutes, VSV-Plexin-B1 was immunoprecipitated (IP) using an anti-VSV antibody and precipitates were immunoblotted (IB) using anti-ErbB-2, anti-VSV or anti-HA antibodies. Shown are the autoluminograms of immunoblots stained with the indicated antibodies. ( B ) MCF-7 cells were incubated with buffer (−) or IKK inhibitor SC-514 (50 µM) for 30 minutes. Thereafter, cells were stimulated without (−) or with 150 nM Sema4D (+) for 20 minutes, lysed, and endogenous Plexin-B1 was immunoprecipitated using an anti-Plexin-B1 antibody. Shown are Western blots of lysed or immunoprecipitated (IP) samples stained with the indicated antibodies (IB). ( C ) 48 hours after transfection with cDNAs encoding truncated versions of VSV-Plexin-B1 (VSV-PlxnB1ΔIC) and HA-ErbB-2 (HA-ErbB-2ΔIC), HEK293 cells were treated without (−) or with IKK inhibitor – SC-514 (50 µM) or NBDBP (100 µM) for 30 minutes. Thereafter, cells were incubated in the absence (−) or presence (+) of 150 nM Sema4D for 20 minutes, lysed and immunoprecipitated (IP) using an anti-VSV antibody. Precipitates were seperated by SDS-PAGE and analyzed by immunoblotting (IB) with anti-VSV- or anti-HA- antibodies. ( D ) MDA-MB-468 cells were incubated without (−) or with SC-514 (50 µM) or NBDBP (100 µM) for 30 minutes, and Plexin-B1 was then immunoprecipitated (IP) from lysed cells. Precipitates were analysed by SDS-PAGE and immunoblotted with antibodies against c-Met or Plexin-B1. Shown are representative examples of at least three experiments.

    Journal: PLoS ONE

    Article Title: The IκB Kinase Complex Is Required for Plexin-B-Mediated Activation of RhoA

    doi: 10.1371/journal.pone.0105661

    Figure Lengend Snippet: ( A ) HEK-293 cells were transfected with cDNAs encoding VSV-Plexin-B1 (VSV-PlxnB1), FLAG-PDZ-RhoGEF (FLAG-PRG) alone or together with a HA-tagged kinase-deficient IKKα-mutant (HA-IKKα-KD). After incubation in the absence (−) or presence (+) of 150 nM Sema4D for 20 minutes, VSV-Plexin-B1 was immunoprecipitated (IP) using an anti-VSV antibody and precipitates were immunoblotted (IB) using anti-ErbB-2, anti-VSV or anti-HA antibodies. Shown are the autoluminograms of immunoblots stained with the indicated antibodies. ( B ) MCF-7 cells were incubated with buffer (−) or IKK inhibitor SC-514 (50 µM) for 30 minutes. Thereafter, cells were stimulated without (−) or with 150 nM Sema4D (+) for 20 minutes, lysed, and endogenous Plexin-B1 was immunoprecipitated using an anti-Plexin-B1 antibody. Shown are Western blots of lysed or immunoprecipitated (IP) samples stained with the indicated antibodies (IB). ( C ) 48 hours after transfection with cDNAs encoding truncated versions of VSV-Plexin-B1 (VSV-PlxnB1ΔIC) and HA-ErbB-2 (HA-ErbB-2ΔIC), HEK293 cells were treated without (−) or with IKK inhibitor – SC-514 (50 µM) or NBDBP (100 µM) for 30 minutes. Thereafter, cells were incubated in the absence (−) or presence (+) of 150 nM Sema4D for 20 minutes, lysed and immunoprecipitated (IP) using an anti-VSV antibody. Precipitates were seperated by SDS-PAGE and analyzed by immunoblotting (IB) with anti-VSV- or anti-HA- antibodies. ( D ) MDA-MB-468 cells were incubated without (−) or with SC-514 (50 µM) or NBDBP (100 µM) for 30 minutes, and Plexin-B1 was then immunoprecipitated (IP) from lysed cells. Precipitates were analysed by SDS-PAGE and immunoblotted with antibodies against c-Met or Plexin-B1. Shown are representative examples of at least three experiments.

    Article Snippet: The following antibodies were used and obtained from commercial sources: Mouse monoclonal anti-ErbB-2 (Invitrogen, 1∶1000), rabbit polyclonal anti-phospho-ErbB-2 (Y1248, 1∶400), rabbit monoclonal anti-RhoA (1∶400), rabbit polyclonal anti-R-Ras (1:400) and rabbit monoclonal anti-IκBα (Cell Signalling Technology, 1:400), mouse monoclonal anti-HA and mouse monoclonal anti-α-tubulin (Sigma-Aldrich, 1∶1000), goat monoclonal anti-Plexin-B1 (R&D Systems, 1∶400), rabbit polyclonal anti-IKKα/β (Santa Cruz Biotechnology, 1∶500), goat polyclonal anti-VSV-G (Thermo Scientific, 1∶1000), mouse monoclonal anti-Met (Invitrogen, 1∶1000).

    Techniques: Transfection, Mutagenesis, Incubation, Immunoprecipitation, Western Blot, Staining, SDS Page

    SHP-1 binds to EGFR and HER-2 protein. Notes: ( A , B ) SKBR3 cells were transfected with control (NC), SHP-1 WT, or SHP-1 MT vector for 48 hours. EGFR and HER-2 proteins were pulled down by immunoprecipitation and the protein level was detected by immunoblotting. ( C , D ) SKBR3 cells were transfected with control (NC), SHP-1 WT, or SHP-1 MT vector for 48 hours. Phosphorylated-EGFR (pEGFR) and phosphorylated-HER-2 (pHER-2) proteins were pulled down by immunoprecipitation and the protein level was detected by immunoblotting. ( E , F ) The binding between SHP-1 and HER-3 or HER-4 was also evaluated through immunoprecipitation. The whole gel is shown, and no specific band of HER-3 or HER-4 was detected. ( G ) Bimolecular fluorescence complementation (BiFC) was performed to confirm the binding activity between SHP-1 and EGFR, HER-2, HER-3, and HER-4. BiFC signals were generated by interaction of the GFP fluorophore components based on proximity. + and − indicate receiving and withholding of the treatment, respectively. Abbreviations: EGFR, epidermal growth factor receptor; HER-2, human epidermal receptor; NC, nonsense control; WT, wild-type; IP, immunoprecipitation.

    Journal: OncoTargets and therapy

    Article Title: Protein tyrosine phosphatase SHP-1 sensitizes EGFR/HER-2 positive breast cancer cells to trastuzumab through modulating phosphorylation of EGFR and HER-2

    doi: 10.2147/OTT.S82225

    Figure Lengend Snippet: SHP-1 binds to EGFR and HER-2 protein. Notes: ( A , B ) SKBR3 cells were transfected with control (NC), SHP-1 WT, or SHP-1 MT vector for 48 hours. EGFR and HER-2 proteins were pulled down by immunoprecipitation and the protein level was detected by immunoblotting. ( C , D ) SKBR3 cells were transfected with control (NC), SHP-1 WT, or SHP-1 MT vector for 48 hours. Phosphorylated-EGFR (pEGFR) and phosphorylated-HER-2 (pHER-2) proteins were pulled down by immunoprecipitation and the protein level was detected by immunoblotting. ( E , F ) The binding between SHP-1 and HER-3 or HER-4 was also evaluated through immunoprecipitation. The whole gel is shown, and no specific band of HER-3 or HER-4 was detected. ( G ) Bimolecular fluorescence complementation (BiFC) was performed to confirm the binding activity between SHP-1 and EGFR, HER-2, HER-3, and HER-4. BiFC signals were generated by interaction of the GFP fluorophore components based on proximity. + and − indicate receiving and withholding of the treatment, respectively. Abbreviations: EGFR, epidermal growth factor receptor; HER-2, human epidermal receptor; NC, nonsense control; WT, wild-type; IP, immunoprecipitation.

    Article Snippet: All antibodies were purchased from Cell Signaling Technologies: phospho-HER2 (Tyr1248) rabbit polyclonal antibody (#2247), HER2 rabbit polyclonal antibody (#2242), phospho-EGFR (Tyr1173) rabbit monoclonal antibody (#4407), EGFR rabbit monoclonal antibody (#4405), phospho-Erk1/2 (Thr202/Tyr204) rabbit monoclonal antibody (#4370), Erk1/2 rabbit monoclonal antibody (#4695), phospho-Stat3 (Tyr705) rabbit monoclonal antibody (#9145), Stat3 rabbit monoclonal antibody (#4904), phospho-Akt (Ser473) rabbit monoclonal antibody (#4060), and Akt (pan) rabbit monoclonal antibody (#4685).

    Techniques: Transfection, Plasmid Preparation, Immunoprecipitation, Western Blot, Binding Assay, Fluorescence, Activity Assay, Generated

    (A) Chromatograms of BT474 cellular metabolites extracted from control and lapatinib (1 μM) treated cells for 2 hours at 37°C. Representative of three independent experiments, all of which showed similar results. (B) Cr kinase enzyme activity assays from the fractionated lysates of BT474 cells treated with vehicle control or 1 μM lapatinib for 2 hours at 37°C. The corresponding immunoblotting (WB) results of the fractionated samples with antibodies against MtCK1, anti-phospho-tyrosine (pTyr), PDHK1 (mitochondria marker), and β-actin (cytosol marker) are also shown. (C) Top: Immunoblots of lysates from 293T cells expressing vector control (−) and HER2 WT. Bottom: qRT-PCR results of MtCK1 mRNA in 293T cells expressing vector control (−) or HER2 WT. (D) Immunoblots of anti-Flag immunoprecipitates from cells expressing Flag-MtCK1 WT and Y153F mutant. (E) Immunoblots of cells treated with 1 μM lapatinib for 2 hours at 37°C. MtCK1 total protein levels were normalized between the control and lapatinib-treated samples to compare the levels of phospho-Y153 MtCK1 between the samples. All results are representative experiments of three independent replicates and P values were determined by a two-tailed Student’s t test (**P<0.01).

    Journal: Cell metabolism

    Article Title: Tyrosine Phosphorylation of Mitochondrial Creatine Kinase 1 Enhances a Druggable Tumor Energy Shuttle Pathway

    doi: 10.1016/j.cmet.2018.08.008

    Figure Lengend Snippet: (A) Chromatograms of BT474 cellular metabolites extracted from control and lapatinib (1 μM) treated cells for 2 hours at 37°C. Representative of three independent experiments, all of which showed similar results. (B) Cr kinase enzyme activity assays from the fractionated lysates of BT474 cells treated with vehicle control or 1 μM lapatinib for 2 hours at 37°C. The corresponding immunoblotting (WB) results of the fractionated samples with antibodies against MtCK1, anti-phospho-tyrosine (pTyr), PDHK1 (mitochondria marker), and β-actin (cytosol marker) are also shown. (C) Top: Immunoblots of lysates from 293T cells expressing vector control (−) and HER2 WT. Bottom: qRT-PCR results of MtCK1 mRNA in 293T cells expressing vector control (−) or HER2 WT. (D) Immunoblots of anti-Flag immunoprecipitates from cells expressing Flag-MtCK1 WT and Y153F mutant. (E) Immunoblots of cells treated with 1 μM lapatinib for 2 hours at 37°C. MtCK1 total protein levels were normalized between the control and lapatinib-treated samples to compare the levels of phospho-Y153 MtCK1 between the samples. All results are representative experiments of three independent replicates and P values were determined by a two-tailed Student’s t test (**P<0.01).

    Article Snippet: Rabbit polyclonal anti-phospho-HER2/ErbB2 (Tyr1248) antibody , Cell Signaling Technology , 2247S, RRID:AB_331725.

    Techniques: Activity Assay, Western Blot, Marker, Expressing, Plasmid Preparation, Quantitative RT-PCR, Mutagenesis, Two Tailed Test

    (A) Top: Immunoblots of lysates from 293T cells co-expressing Flag-MtCK1 WT or Y153F along with vector control (−) or HER2 WT. Bottom: Densitometric quantification of four independent anti-Flag and anti-β-actin immunoblots based on the densitometric analysis showing a dose-dependent linear correlation of anti-Flag and anti-β-actin immunoblots of the lysates (Figure S2A). (B) Top: Immunoblots of SKBR3 cells stably expressing Flag-MtCK1 WT or Y153F that were treated with 200 μg/ml cycloheximide (CHX) for the indicated times. Bottom: Densitometric quantification of three independent anti-Flag and anti-β-actin immunoblots. (C) Left: MtCK1 enzyme activity in anti-Flag immunoprecipitates from 293T cells co-expressing Flag-MtCK1 WT, MtCK1 Y153F or MtCK1 E227L and HER2. Right: Immunoblots of the corresponding anti-Flag immunoprecipitates (IP) and whole-cell lysates (WCL) from 293T cells. (D) Immunoblots of 293T cells co-expressing either vector control (−) or HER2 and Flag-MtCK1 E227L. All results are representative experiments of three independent replicates. P values were determined by a two-tailed Student’s t test (*P<0.05, **P<0.01, ***p < 0.001).

    Journal: Cell metabolism

    Article Title: Tyrosine Phosphorylation of Mitochondrial Creatine Kinase 1 Enhances a Druggable Tumor Energy Shuttle Pathway

    doi: 10.1016/j.cmet.2018.08.008

    Figure Lengend Snippet: (A) Top: Immunoblots of lysates from 293T cells co-expressing Flag-MtCK1 WT or Y153F along with vector control (−) or HER2 WT. Bottom: Densitometric quantification of four independent anti-Flag and anti-β-actin immunoblots based on the densitometric analysis showing a dose-dependent linear correlation of anti-Flag and anti-β-actin immunoblots of the lysates (Figure S2A). (B) Top: Immunoblots of SKBR3 cells stably expressing Flag-MtCK1 WT or Y153F that were treated with 200 μg/ml cycloheximide (CHX) for the indicated times. Bottom: Densitometric quantification of three independent anti-Flag and anti-β-actin immunoblots. (C) Left: MtCK1 enzyme activity in anti-Flag immunoprecipitates from 293T cells co-expressing Flag-MtCK1 WT, MtCK1 Y153F or MtCK1 E227L and HER2. Right: Immunoblots of the corresponding anti-Flag immunoprecipitates (IP) and whole-cell lysates (WCL) from 293T cells. (D) Immunoblots of 293T cells co-expressing either vector control (−) or HER2 and Flag-MtCK1 E227L. All results are representative experiments of three independent replicates. P values were determined by a two-tailed Student’s t test (*P<0.05, **P<0.01, ***p < 0.001).

    Article Snippet: Rabbit polyclonal anti-phospho-HER2/ErbB2 (Tyr1248) antibody , Cell Signaling Technology , 2247S, RRID:AB_331725.

    Techniques: Western Blot, Expressing, Plasmid Preparation, Stable Transfection, Activity Assay, Two Tailed Test

    (A) Immunoblots of lysates (left) and qRT-PCR for MtCK1 mRNA (right) from “rescued” BT474 cells that stably express Flag-MtCK1 WT or Y153F with stable knockdown of endogenous MtCK1. (B) Immunoblots of “rescued” BT474 cells expressing Flag-MtCK1 WT and Y153F after treatment with MG132 for 6 h. (C) Immunoblots of anti-Flag immunoprecipitates and whole-cell lysates (WCL) from Flag-MtCK1 WT and Y153F “rescued” BT474 cells. (D) Immunoblots (WB) of anti-TRAP1 or anti-IgG immunoprecipitates and whole-cell lysates (WCL) from BT474 cells with or without lapatinib treatment (500 nM, 6 h). (E) Immunoblots (WB) of anti-Flag immunoprecipitates and WCL from 293T cells expressing Flag-MtCK1 WT expressing control vector (−) or HER2. (F) Immunoblots of lysates (left) and PCr levels (right) from 293T cells expressing vector control (−) or HER2 with or without stable knockdown of endogenous TRAP1. All results are representative experiments of three independent replicates. P values were determined by a two-tailed Student’s t test (*P<0.05).

    Journal: Cell metabolism

    Article Title: Tyrosine Phosphorylation of Mitochondrial Creatine Kinase 1 Enhances a Druggable Tumor Energy Shuttle Pathway

    doi: 10.1016/j.cmet.2018.08.008

    Figure Lengend Snippet: (A) Immunoblots of lysates (left) and qRT-PCR for MtCK1 mRNA (right) from “rescued” BT474 cells that stably express Flag-MtCK1 WT or Y153F with stable knockdown of endogenous MtCK1. (B) Immunoblots of “rescued” BT474 cells expressing Flag-MtCK1 WT and Y153F after treatment with MG132 for 6 h. (C) Immunoblots of anti-Flag immunoprecipitates and whole-cell lysates (WCL) from Flag-MtCK1 WT and Y153F “rescued” BT474 cells. (D) Immunoblots (WB) of anti-TRAP1 or anti-IgG immunoprecipitates and whole-cell lysates (WCL) from BT474 cells with or without lapatinib treatment (500 nM, 6 h). (E) Immunoblots (WB) of anti-Flag immunoprecipitates and WCL from 293T cells expressing Flag-MtCK1 WT expressing control vector (−) or HER2. (F) Immunoblots of lysates (left) and PCr levels (right) from 293T cells expressing vector control (−) or HER2 with or without stable knockdown of endogenous TRAP1. All results are representative experiments of three independent replicates. P values were determined by a two-tailed Student’s t test (*P<0.05).

    Article Snippet: Rabbit polyclonal anti-phospho-HER2/ErbB2 (Tyr1248) antibody , Cell Signaling Technology , 2247S, RRID:AB_331725.

    Techniques: Western Blot, Quantitative RT-PCR, Stable Transfection, Expressing, Plasmid Preparation, Two Tailed Test

    (A) Immunoblots of an in vitro tyrosine kinase assay using recombinant ABL with purified recombinant MtCK1 WT protein. (B) Immunoblots of in vitro ABL kinase assay using recombinant ABL and MtCK1 WT or Y153F proteins. (C) Immunoblots of anti-Flag immunoprecipitates and WCL from 293T cells co-expressing Flag-MtCK1 WT and vector control (−), HER2 WT or ABL. (D) Immunoblots of BT474 cells treated with DMSO, or imatinib (20 βM), or lapatinib (500 nM) for 6 h. (E) Immunoblots of anti-Flag immunoprecipitates and WCL from BT474 cells treated with imatinib (10 βM) overnight. (F) Immunoblots of anti-TRAP1 or anti-IgG immunoprecipitates and WCL from BT474 cells with or without imatinib treatment (20 βM) for 6 h. (G) Immunoblots of anti-IgG control and anti-MtCK1 antibody immunoprecipitates and input lysate from BT474 cells. (H) Isolated mitochondria from BT474 cells were treated with 5 μg/mL proteinase K in the absence and presence of 1% Triton X-100 and immunoblotted for the indicated proteins. All immunoblots results are representative experiments of three independent replicates.

    Journal: Cell metabolism

    Article Title: Tyrosine Phosphorylation of Mitochondrial Creatine Kinase 1 Enhances a Druggable Tumor Energy Shuttle Pathway

    doi: 10.1016/j.cmet.2018.08.008

    Figure Lengend Snippet: (A) Immunoblots of an in vitro tyrosine kinase assay using recombinant ABL with purified recombinant MtCK1 WT protein. (B) Immunoblots of in vitro ABL kinase assay using recombinant ABL and MtCK1 WT or Y153F proteins. (C) Immunoblots of anti-Flag immunoprecipitates and WCL from 293T cells co-expressing Flag-MtCK1 WT and vector control (−), HER2 WT or ABL. (D) Immunoblots of BT474 cells treated with DMSO, or imatinib (20 βM), or lapatinib (500 nM) for 6 h. (E) Immunoblots of anti-Flag immunoprecipitates and WCL from BT474 cells treated with imatinib (10 βM) overnight. (F) Immunoblots of anti-TRAP1 or anti-IgG immunoprecipitates and WCL from BT474 cells with or without imatinib treatment (20 βM) for 6 h. (G) Immunoblots of anti-IgG control and anti-MtCK1 antibody immunoprecipitates and input lysate from BT474 cells. (H) Isolated mitochondria from BT474 cells were treated with 5 μg/mL proteinase K in the absence and presence of 1% Triton X-100 and immunoblotted for the indicated proteins. All immunoblots results are representative experiments of three independent replicates.

    Article Snippet: Rabbit polyclonal anti-phospho-HER2/ErbB2 (Tyr1248) antibody , Cell Signaling Technology , 2247S, RRID:AB_331725.

    Techniques: Western Blot, In Vitro, Tyrosine Kinase Assay, Recombinant, Purification, Kinase Assay, Expressing, Plasmid Preparation, Isolation

    (A) Immunoblots of three normal breast tissue lysates (#1-#3), six HER2+ patient-derived xenograft (PDX) tumor lysates (#4-#9), five TNBC PDX tumor lysates (#10-#14). (B) Tumor volumes in NSG mice (N=5) with PDX #4 cells with or without MtCK1 knockdown. Each dot represents tumor volume in an individual mouse with error bars ± standard deviation. Immunoblots of lysates from the corresponding cells are also shown. (C) Structures of Cr and Cyclo-Cr. (D) HPLC analysis of metabolites extracted from SKBR3-R cells treated with either water or Cyclo-Cr (1 mM) overnight. The experiment shown is representative of three independent experiments, all of which showed similar results. (E) Clonogenic assay results of breast cancer cell lines treated with Cyclo-Cr. (F) Cell proliferation assay results of SKBR3-R treated with Cyclo-Cr and/or PCr. (G) Cell proliferation assay results of normal primary mammary epithelial cells up to 6 mM of CCr. Error bars in E-G ± standard deviation of 3 independent measurements. P values were determined by a two-tailed Student’s t test. ***P<0.001.

    Journal: Cell metabolism

    Article Title: Tyrosine Phosphorylation of Mitochondrial Creatine Kinase 1 Enhances a Druggable Tumor Energy Shuttle Pathway

    doi: 10.1016/j.cmet.2018.08.008

    Figure Lengend Snippet: (A) Immunoblots of three normal breast tissue lysates (#1-#3), six HER2+ patient-derived xenograft (PDX) tumor lysates (#4-#9), five TNBC PDX tumor lysates (#10-#14). (B) Tumor volumes in NSG mice (N=5) with PDX #4 cells with or without MtCK1 knockdown. Each dot represents tumor volume in an individual mouse with error bars ± standard deviation. Immunoblots of lysates from the corresponding cells are also shown. (C) Structures of Cr and Cyclo-Cr. (D) HPLC analysis of metabolites extracted from SKBR3-R cells treated with either water or Cyclo-Cr (1 mM) overnight. The experiment shown is representative of three independent experiments, all of which showed similar results. (E) Clonogenic assay results of breast cancer cell lines treated with Cyclo-Cr. (F) Cell proliferation assay results of SKBR3-R treated with Cyclo-Cr and/or PCr. (G) Cell proliferation assay results of normal primary mammary epithelial cells up to 6 mM of CCr. Error bars in E-G ± standard deviation of 3 independent measurements. P values were determined by a two-tailed Student’s t test. ***P<0.001.

    Article Snippet: Rabbit polyclonal anti-phospho-HER2/ErbB2 (Tyr1248) antibody , Cell Signaling Technology , 2247S, RRID:AB_331725.

    Techniques: Western Blot, Derivative Assay, Standard Deviation, Clonogenic Assay, Proliferation Assay, Two Tailed Test

    (A) Tumor growth in nude mice with BT474-R tumors treated with 0.3% Cyclo-Cr in drinking water versus regular drinking water. The Cyclo-Cr and PCr concentrations in the harvested BT474-R tumors determined by HPLC analysis are also shown below. N=8 in each group. (B) Tumor growth of HER2+ PDX #4 in NSG mice treated with 0.3% Cyclo-Cr in drinking water versus regular drinking water. The Cyclo-Cr and PCr concentrations in harvested PDX #4 tumors determined by HPLC analysis are also shown below. N=6 in each group. (C) Left: Tumor growth in NSG mice with trastuzumab-resistant HER2+ PDX #4 under various drug combinations (a total of 6 groups; control, Cyclo-Cr alone, lapatinib alone, Cyclo-Cr plus PCr, Cyclo-Cr plus lapatinib, and Cyclo-Cr plus lapatinib plus PCr). Cyclo-Cr was administered in drinking water at 0.3%, lapatinib was administrated orally at 50 mg/kg/day and PCr was administered at 400 mg/kg/day intraperitoneally. Right: Bar graphs represent tumor volume in mice with various treatments at day 20. N=9 in each group. P values were determined by a two-tailed Student’s t test. *P<0.05, **P<0.01, ***P<0.001.

    Journal: Cell metabolism

    Article Title: Tyrosine Phosphorylation of Mitochondrial Creatine Kinase 1 Enhances a Druggable Tumor Energy Shuttle Pathway

    doi: 10.1016/j.cmet.2018.08.008

    Figure Lengend Snippet: (A) Tumor growth in nude mice with BT474-R tumors treated with 0.3% Cyclo-Cr in drinking water versus regular drinking water. The Cyclo-Cr and PCr concentrations in the harvested BT474-R tumors determined by HPLC analysis are also shown below. N=8 in each group. (B) Tumor growth of HER2+ PDX #4 in NSG mice treated with 0.3% Cyclo-Cr in drinking water versus regular drinking water. The Cyclo-Cr and PCr concentrations in harvested PDX #4 tumors determined by HPLC analysis are also shown below. N=6 in each group. (C) Left: Tumor growth in NSG mice with trastuzumab-resistant HER2+ PDX #4 under various drug combinations (a total of 6 groups; control, Cyclo-Cr alone, lapatinib alone, Cyclo-Cr plus PCr, Cyclo-Cr plus lapatinib, and Cyclo-Cr plus lapatinib plus PCr). Cyclo-Cr was administered in drinking water at 0.3%, lapatinib was administrated orally at 50 mg/kg/day and PCr was administered at 400 mg/kg/day intraperitoneally. Right: Bar graphs represent tumor volume in mice with various treatments at day 20. N=9 in each group. P values were determined by a two-tailed Student’s t test. *P<0.05, **P<0.01, ***P<0.001.

    Article Snippet: Rabbit polyclonal anti-phospho-HER2/ErbB2 (Tyr1248) antibody , Cell Signaling Technology , 2247S, RRID:AB_331725.

    Techniques: Two Tailed Test

    KEY RESOURCES TABLE

    Journal: Cell metabolism

    Article Title: Tyrosine Phosphorylation of Mitochondrial Creatine Kinase 1 Enhances a Druggable Tumor Energy Shuttle Pathway

    doi: 10.1016/j.cmet.2018.08.008

    Figure Lengend Snippet: KEY RESOURCES TABLE

    Article Snippet: Rabbit polyclonal anti-phospho-HER2/ErbB2 (Tyr1248) antibody , Cell Signaling Technology , 2247S, RRID:AB_331725.

    Techniques: Derivative Assay, Recombinant, FLAG-tag, Plasmid Preparation, shRNA, Software, Clone Assay, Mutagenesis, SYBR Green Assay, CyQUANT Assay, Proliferation Assay, Cell Viability Assay, Protease Inhibitor

    (A) Sequences of the TM and JM regions of WT EGFR and ErbB2. GXXXG motifs are noted with orange lines. Conserved residues are highlighted in yellow. JM residues substituted with Cys in CCH-1 or CCH-10 ErbB2 are indicated with green (M663 and L667) or blue dots (R665 and Q668), respectively. (B) Representative TIRF-M images of CHO-K1 cells expressing FLAG-tagged WT or V635G CCH-1 and CCH-10 ErbB2 after ReAsH treatment. Scale bars represent 10 μm. (C and D) Quantification of TIRF-M results, from “n” number of cells, as a fold increase in expression-corrected ReAsH fluorescence over background of CHO-K1 cells expressing WT, CCH-1, or CCH-10 ErbB2 (with/without a V635G mutation in the TM domain). Error bars represent SEM. ***p<0.001 and ****p<0.0001 from one-way ANOVA with Bonferroni post-analysis accounting for multiple comparisons. See also Figure S6 and Table S1.

    Journal: Cell chemical biology

    Article Title: Mechanism of allosteric coupling into and through the plasma membrane by EGFR

    doi: 10.1016/j.chembiol.2018.04.005

    Figure Lengend Snippet: (A) Sequences of the TM and JM regions of WT EGFR and ErbB2. GXXXG motifs are noted with orange lines. Conserved residues are highlighted in yellow. JM residues substituted with Cys in CCH-1 or CCH-10 ErbB2 are indicated with green (M663 and L667) or blue dots (R665 and Q668), respectively. (B) Representative TIRF-M images of CHO-K1 cells expressing FLAG-tagged WT or V635G CCH-1 and CCH-10 ErbB2 after ReAsH treatment. Scale bars represent 10 μm. (C and D) Quantification of TIRF-M results, from “n” number of cells, as a fold increase in expression-corrected ReAsH fluorescence over background of CHO-K1 cells expressing WT, CCH-1, or CCH-10 ErbB2 (with/without a V635G mutation in the TM domain). Error bars represent SEM. ***p<0.001 and ****p<0.0001 from one-way ANOVA with Bonferroni post-analysis accounting for multiple comparisons. See also Figure S6 and Table S1.

    Article Snippet: Rabbit polyclonal anti-Phospho-Her2/ErbB2 Tyr 1248 , Cell Signaling Technology , Cat#2247; RRID: AB_331725.

    Techniques: Expressing, Fluorescence, Mutagenesis

    KEY RESOURCES TABLE

    Journal: Cell chemical biology

    Article Title: Mechanism of allosteric coupling into and through the plasma membrane by EGFR

    doi: 10.1016/j.chembiol.2018.04.005

    Figure Lengend Snippet: KEY RESOURCES TABLE

    Article Snippet: Rabbit polyclonal anti-Phospho-Her2/ErbB2 Tyr 1248 , Cell Signaling Technology , Cat#2247; RRID: AB_331725.

    Techniques: Recombinant, Protease Inhibitor, Binding Assay, Western Blot, Mutagenesis, Fluorescence, Live Cell Imaging, Software

    Journal: Cell Systems

    Article Title: Context Specificity in Causal Signaling Networks Revealed by Phosphoprotein Profiling

    doi: 10.1016/j.cels.2016.11.013

    Figure Lengend Snippet:

    Article Snippet: Rabbit polyclonal anti-phospho-EGFR_pY992 , Cell Signaling Technology , Cat#2235; RRID: AB_331709.

    Techniques: Transduction, Recombinant, Protease Inhibitor, Bicinchoninic Acid Protein Assay, Software, Inhibition, Modification

    A. Left panel , visualization of the interaction between ErbB2 and nucleolin (red dots) in naïve MDCK cells and MDCK ErbB2-expressing clones was performed using a proximity ligation assay (PLA). Right panel , differences between signal intensity in both cell lines represented as the number of dots per cell (means ±SE). B. Left panel , visualization of the interaction between ErbB2 and nucleolin (red dots) in SKBR3 untreated cells and cells treated with either control siRNA or ErbB2-specific siRNA. Upper right panel , a western-blot analysis of ErbB2-siRNA treatment effect on ErbB2 levels in SKBR3 cells; numbers below bands indicate average fold induction of untreated. Lower right panel , differences in signal intensity represented as number of dots per cell (means ±SE).

    Journal: Oncotarget

    Article Title: Nucleolin-binding by ErbB2 enhances tumorigenicity of ErbB2-positive breast cancer

    doi: 10.18632/oncotarget.11323

    Figure Lengend Snippet: A. Left panel , visualization of the interaction between ErbB2 and nucleolin (red dots) in naïve MDCK cells and MDCK ErbB2-expressing clones was performed using a proximity ligation assay (PLA). Right panel , differences between signal intensity in both cell lines represented as the number of dots per cell (means ±SE). B. Left panel , visualization of the interaction between ErbB2 and nucleolin (red dots) in SKBR3 untreated cells and cells treated with either control siRNA or ErbB2-specific siRNA. Upper right panel , a western-blot analysis of ErbB2-siRNA treatment effect on ErbB2 levels in SKBR3 cells; numbers below bands indicate average fold induction of untreated. Lower right panel , differences in signal intensity represented as number of dots per cell (means ±SE).

    Article Snippet: The antibodies used are as follows: monoclonal mouse anti-actin (691001; MP Biomedicals, Santa Ana, CA); polyclonal rabbit anti-ErbB2 (HER2/neu), rabbit anti-nucleolin (C23), rabbit anti-Erk2 and rabbit anti-Akt, monoclonal mouse anti-phosphotyrosine (pY20) and monoclonal mouse anti-GFP and mouse anti-nucleolin (sc-284; sc-13057; sc-154; sc-8312; sc-508; sc-9996; sc-8031, respectively; Santa Cruz Biotechnology, Dallas, TX); monoclonal mouse anti-tubulin (T7816; Sigma-Aldrich); polyclonal rabbit anti-phospho-ErbB2, rabbit anti-phospho-Akt, rabbit anti-phospho-Erk1/2 (2249; 4058; 9101, respectively; Cell Signaling Technology, Danvers, MA); and monoclonal mouse anti-ErbB2 (extracellular; L87), which was a gift from Prof. Y. Yarden, Weizmann Institute of Science, Israel.

    Techniques: Expressing, Clone Assay, Proximity Ligation Assay, Western Blot

    A. Colony formation was tested in either naïve MDCK cells or MDCK stable clones expressing ErbB2 and nucleolin, either separately or in combination. Upper panel , representative images. Lower panel , total colonies area presented as fold induction of control (means ±SD). B. MDCK cells overexpressing nucleolin, ErbB2, nucleolin and ErbB2 or none were plated and grown in soft agar, as described in Materials and Methods. Upper panel , photomicrographs of typical wells. Lower panel , number of colonies (size>0.01 mm 2 ; means ±SD). *** ( p-value <0.005) - ErbB2 and nucleolin clone compared to the controls; ^^^ ( p-value <0.005) - ErbB2/nucleolin clones compared to naïve control.

    Journal: Oncotarget

    Article Title: Nucleolin-binding by ErbB2 enhances tumorigenicity of ErbB2-positive breast cancer

    doi: 10.18632/oncotarget.11323

    Figure Lengend Snippet: A. Colony formation was tested in either naïve MDCK cells or MDCK stable clones expressing ErbB2 and nucleolin, either separately or in combination. Upper panel , representative images. Lower panel , total colonies area presented as fold induction of control (means ±SD). B. MDCK cells overexpressing nucleolin, ErbB2, nucleolin and ErbB2 or none were plated and grown in soft agar, as described in Materials and Methods. Upper panel , photomicrographs of typical wells. Lower panel , number of colonies (size>0.01 mm 2 ; means ±SD). *** ( p-value <0.005) - ErbB2 and nucleolin clone compared to the controls; ^^^ ( p-value <0.005) - ErbB2/nucleolin clones compared to naïve control.

    Article Snippet: The antibodies used are as follows: monoclonal mouse anti-actin (691001; MP Biomedicals, Santa Ana, CA); polyclonal rabbit anti-ErbB2 (HER2/neu), rabbit anti-nucleolin (C23), rabbit anti-Erk2 and rabbit anti-Akt, monoclonal mouse anti-phosphotyrosine (pY20) and monoclonal mouse anti-GFP and mouse anti-nucleolin (sc-284; sc-13057; sc-154; sc-8312; sc-508; sc-9996; sc-8031, respectively; Santa Cruz Biotechnology, Dallas, TX); monoclonal mouse anti-tubulin (T7816; Sigma-Aldrich); polyclonal rabbit anti-phospho-ErbB2, rabbit anti-phospho-Akt, rabbit anti-phospho-Erk1/2 (2249; 4058; 9101, respectively; Cell Signaling Technology, Danvers, MA); and monoclonal mouse anti-ErbB2 (extracellular; L87), which was a gift from Prof. Y. Yarden, Weizmann Institute of Science, Israel.

    Techniques: Clone Assay, Expressing

    A. Schematic representation of full-length (wt) nucleolin and its truncation variants: N-ter, 212, RBD and GAR. B. Co-immunoprecipitation (co-IP) analysis of ErbB2 and full-length nucleolin (NCL), N-ter and 212. HEK-293T cells were co-transfected with ErbB2 and either nucleolin variants or control vectors. C. Co-IP analysis of ErbB2 and the C-terminal variants of nucleolin: 212, RBD and GAR. HEK-293T cells were co-transfected with ErbB2 and either 212 (as a positive control), RBD, GAR or control vectors.

    Journal: Oncotarget

    Article Title: Nucleolin-binding by ErbB2 enhances tumorigenicity of ErbB2-positive breast cancer

    doi: 10.18632/oncotarget.11323

    Figure Lengend Snippet: A. Schematic representation of full-length (wt) nucleolin and its truncation variants: N-ter, 212, RBD and GAR. B. Co-immunoprecipitation (co-IP) analysis of ErbB2 and full-length nucleolin (NCL), N-ter and 212. HEK-293T cells were co-transfected with ErbB2 and either nucleolin variants or control vectors. C. Co-IP analysis of ErbB2 and the C-terminal variants of nucleolin: 212, RBD and GAR. HEK-293T cells were co-transfected with ErbB2 and either 212 (as a positive control), RBD, GAR or control vectors.

    Article Snippet: The antibodies used are as follows: monoclonal mouse anti-actin (691001; MP Biomedicals, Santa Ana, CA); polyclonal rabbit anti-ErbB2 (HER2/neu), rabbit anti-nucleolin (C23), rabbit anti-Erk2 and rabbit anti-Akt, monoclonal mouse anti-phosphotyrosine (pY20) and monoclonal mouse anti-GFP and mouse anti-nucleolin (sc-284; sc-13057; sc-154; sc-8312; sc-508; sc-9996; sc-8031, respectively; Santa Cruz Biotechnology, Dallas, TX); monoclonal mouse anti-tubulin (T7816; Sigma-Aldrich); polyclonal rabbit anti-phospho-ErbB2, rabbit anti-phospho-Akt, rabbit anti-phospho-Erk1/2 (2249; 4058; 9101, respectively; Cell Signaling Technology, Danvers, MA); and monoclonal mouse anti-ErbB2 (extracellular; L87), which was a gift from Prof. Y. Yarden, Weizmann Institute of Science, Israel.

    Techniques: Immunoprecipitation, Co-Immunoprecipitation Assay, Transfection, Positive Control

    A. Schematic representation of full-length (wt) ErbB2 and its mutants: Δcyt-NLS and ΔNLS. B. Co-IP analysis of nucleolin and ErbB2 mutants. HEK-293T cells were co-transfected with Flag-tagged nucleolin and either wt ErbB2, ΔNLS, Δcyt-NLS or control vectors.

    Journal: Oncotarget

    Article Title: Nucleolin-binding by ErbB2 enhances tumorigenicity of ErbB2-positive breast cancer

    doi: 10.18632/oncotarget.11323

    Figure Lengend Snippet: A. Schematic representation of full-length (wt) ErbB2 and its mutants: Δcyt-NLS and ΔNLS. B. Co-IP analysis of nucleolin and ErbB2 mutants. HEK-293T cells were co-transfected with Flag-tagged nucleolin and either wt ErbB2, ΔNLS, Δcyt-NLS or control vectors.

    Article Snippet: The antibodies used are as follows: monoclonal mouse anti-actin (691001; MP Biomedicals, Santa Ana, CA); polyclonal rabbit anti-ErbB2 (HER2/neu), rabbit anti-nucleolin (C23), rabbit anti-Erk2 and rabbit anti-Akt, monoclonal mouse anti-phosphotyrosine (pY20) and monoclonal mouse anti-GFP and mouse anti-nucleolin (sc-284; sc-13057; sc-154; sc-8312; sc-508; sc-9996; sc-8031, respectively; Santa Cruz Biotechnology, Dallas, TX); monoclonal mouse anti-tubulin (T7816; Sigma-Aldrich); polyclonal rabbit anti-phospho-ErbB2, rabbit anti-phospho-Akt, rabbit anti-phospho-Erk1/2 (2249; 4058; 9101, respectively; Cell Signaling Technology, Danvers, MA); and monoclonal mouse anti-ErbB2 (extracellular; L87), which was a gift from Prof. Y. Yarden, Weizmann Institute of Science, Israel.

    Techniques: Co-Immunoprecipitation Assay, Transfection

    Phosphorylation levels of ErbB2 were determined by western-blotting using an anti-phospho-ErbB2 antibody. A. SKBR3 cancer cells were transiently transfected with anti-nucleolin shRNA; numbers below bands indicate average fold induction of nucleolin relative to untreated cells (means ±SD). B. HEK-293T cells were transiently transfected with ErbB2 and either wt nucleolin, N-ter, 212 or a control vector (means ±SD). C. HEK-293T cells were transiently transfected with ErbB2 or ΔNLS and either wt nucleolin or a control vector (means ±SD; *** ( p-value <0.005) - nucleolin-transfected compared to GFP-transfected control; ^^ ( p-value <0.01) -ΔNLS-transfected compared to wt ErbB2-transfected cells). D. HEK-293T cells were transiently transfected with either ErbB1 alone or in combination with wt ErbB2 or ΔNLS, and total phosphorylation levels of Y20 were measured by western-blotting, prior to and following EGF-stimulation (10min), as indicated (means ±SD; *** ( p-value <0.005) - EGF-stimulated compared to unstimulated cells; ^^^ ( p-value <0.005) - comparison between co-transfections in cells either unstimulated or stimulated with EGF).

    Journal: Oncotarget

    Article Title: Nucleolin-binding by ErbB2 enhances tumorigenicity of ErbB2-positive breast cancer

    doi: 10.18632/oncotarget.11323

    Figure Lengend Snippet: Phosphorylation levels of ErbB2 were determined by western-blotting using an anti-phospho-ErbB2 antibody. A. SKBR3 cancer cells were transiently transfected with anti-nucleolin shRNA; numbers below bands indicate average fold induction of nucleolin relative to untreated cells (means ±SD). B. HEK-293T cells were transiently transfected with ErbB2 and either wt nucleolin, N-ter, 212 or a control vector (means ±SD). C. HEK-293T cells were transiently transfected with ErbB2 or ΔNLS and either wt nucleolin or a control vector (means ±SD; *** ( p-value <0.005) - nucleolin-transfected compared to GFP-transfected control; ^^ ( p-value <0.01) -ΔNLS-transfected compared to wt ErbB2-transfected cells). D. HEK-293T cells were transiently transfected with either ErbB1 alone or in combination with wt ErbB2 or ΔNLS, and total phosphorylation levels of Y20 were measured by western-blotting, prior to and following EGF-stimulation (10min), as indicated (means ±SD; *** ( p-value <0.005) - EGF-stimulated compared to unstimulated cells; ^^^ ( p-value <0.005) - comparison between co-transfections in cells either unstimulated or stimulated with EGF).

    Article Snippet: The antibodies used are as follows: monoclonal mouse anti-actin (691001; MP Biomedicals, Santa Ana, CA); polyclonal rabbit anti-ErbB2 (HER2/neu), rabbit anti-nucleolin (C23), rabbit anti-Erk2 and rabbit anti-Akt, monoclonal mouse anti-phosphotyrosine (pY20) and monoclonal mouse anti-GFP and mouse anti-nucleolin (sc-284; sc-13057; sc-154; sc-8312; sc-508; sc-9996; sc-8031, respectively; Santa Cruz Biotechnology, Dallas, TX); monoclonal mouse anti-tubulin (T7816; Sigma-Aldrich); polyclonal rabbit anti-phospho-ErbB2, rabbit anti-phospho-Akt, rabbit anti-phospho-Erk1/2 (2249; 4058; 9101, respectively; Cell Signaling Technology, Danvers, MA); and monoclonal mouse anti-ErbB2 (extracellular; L87), which was a gift from Prof. Y. Yarden, Weizmann Institute of Science, Israel.

    Techniques: Western Blot, Transfection, shRNA, Plasmid Preparation

    A. Western-blot analysis of activation levels of ErbB2 downstream signaling pathways, as determined by phosphorylation of Erk and Akt proteins in HEK-293T cells transiently transfected with ErbB2, nucleolin, neither or both (means ±SD). B. Activation of ErbB2 by nucleolin and its downstream signaling was inhibited by transient overexpression of GFP-TM-NLS peptides in stably GFP or GFP-nucleolin-overexpressing SKBR3 breast cancer cells (means ±SD). C. SKBR3 cells were transiently transfected with either GFP-TM-NLS or a control, and cell viability was measured using the methylene blue assay (means ±SD).

    Journal: Oncotarget

    Article Title: Nucleolin-binding by ErbB2 enhances tumorigenicity of ErbB2-positive breast cancer

    doi: 10.18632/oncotarget.11323

    Figure Lengend Snippet: A. Western-blot analysis of activation levels of ErbB2 downstream signaling pathways, as determined by phosphorylation of Erk and Akt proteins in HEK-293T cells transiently transfected with ErbB2, nucleolin, neither or both (means ±SD). B. Activation of ErbB2 by nucleolin and its downstream signaling was inhibited by transient overexpression of GFP-TM-NLS peptides in stably GFP or GFP-nucleolin-overexpressing SKBR3 breast cancer cells (means ±SD). C. SKBR3 cells were transiently transfected with either GFP-TM-NLS or a control, and cell viability was measured using the methylene blue assay (means ±SD).

    Article Snippet: The antibodies used are as follows: monoclonal mouse anti-actin (691001; MP Biomedicals, Santa Ana, CA); polyclonal rabbit anti-ErbB2 (HER2/neu), rabbit anti-nucleolin (C23), rabbit anti-Erk2 and rabbit anti-Akt, monoclonal mouse anti-phosphotyrosine (pY20) and monoclonal mouse anti-GFP and mouse anti-nucleolin (sc-284; sc-13057; sc-154; sc-8312; sc-508; sc-9996; sc-8031, respectively; Santa Cruz Biotechnology, Dallas, TX); monoclonal mouse anti-tubulin (T7816; Sigma-Aldrich); polyclonal rabbit anti-phospho-ErbB2, rabbit anti-phospho-Akt, rabbit anti-phospho-Erk1/2 (2249; 4058; 9101, respectively; Cell Signaling Technology, Danvers, MA); and monoclonal mouse anti-ErbB2 (extracellular; L87), which was a gift from Prof. Y. Yarden, Weizmann Institute of Science, Israel.

    Techniques: Western Blot, Activation Assay, Transfection, Over Expression, Stable Transfection

    A. Kaplan-Meier curves depicting survival rates of patients overexpressing either ErbB2 alone or both ErbB2 and nucleolin (n=64). B. Cox-proportional hazards model for ErbB2-positive breast cancer patients, with or without nucleolin overexpression (n=305). C. Age of disease onset in patients overexpressing either ErbB2 alone or both ErbB2 and nucleolin (n=518). D. Time from initial diagnosis until detection of an additional, new, tumor (metastatic or primary) in the same patient (n=42). Data was compared between patients overexpressing ErbB2 and patients overexpressing ErbB2 and nucleolin.

    Journal: Oncotarget

    Article Title: Nucleolin-binding by ErbB2 enhances tumorigenicity of ErbB2-positive breast cancer

    doi: 10.18632/oncotarget.11323

    Figure Lengend Snippet: A. Kaplan-Meier curves depicting survival rates of patients overexpressing either ErbB2 alone or both ErbB2 and nucleolin (n=64). B. Cox-proportional hazards model for ErbB2-positive breast cancer patients, with or without nucleolin overexpression (n=305). C. Age of disease onset in patients overexpressing either ErbB2 alone or both ErbB2 and nucleolin (n=518). D. Time from initial diagnosis until detection of an additional, new, tumor (metastatic or primary) in the same patient (n=42). Data was compared between patients overexpressing ErbB2 and patients overexpressing ErbB2 and nucleolin.

    Article Snippet: The antibodies used are as follows: monoclonal mouse anti-actin (691001; MP Biomedicals, Santa Ana, CA); polyclonal rabbit anti-ErbB2 (HER2/neu), rabbit anti-nucleolin (C23), rabbit anti-Erk2 and rabbit anti-Akt, monoclonal mouse anti-phosphotyrosine (pY20) and monoclonal mouse anti-GFP and mouse anti-nucleolin (sc-284; sc-13057; sc-154; sc-8312; sc-508; sc-9996; sc-8031, respectively; Santa Cruz Biotechnology, Dallas, TX); monoclonal mouse anti-tubulin (T7816; Sigma-Aldrich); polyclonal rabbit anti-phospho-ErbB2, rabbit anti-phospho-Akt, rabbit anti-phospho-Erk1/2 (2249; 4058; 9101, respectively; Cell Signaling Technology, Danvers, MA); and monoclonal mouse anti-ErbB2 (extracellular; L87), which was a gift from Prof. Y. Yarden, Weizmann Institute of Science, Israel.

    Techniques: Over Expression

    A. SKBR3 cells were treated with GroA (10μM), and cell viability was measured by the methylene blue assay at the indicated time points (means ±SD). B. Western-blot analysis of ErbB2 activation, determined by ErbB2 phosphorylation levels, following treatment with GroA (means ±SD). C. SKBR3 cells overexpressing either GFP-nucleolin (SKBR-NCL) or GFP (SKBR-GFP) were treated with GroA (10μM), and cell viability was measured using the methylene blue assay. Left panel , time course curves of cell viability following treatment in SKBR-GFP (control) and SKBR-NCL cells. Right panel , comparison of cell viability in SKBR-GFP and SKBR-NCL cells after 5 days of treatment. Results presented as means ±SD. D. The IC-10, 20 and 50 values for GroA treatment were determined for SKBR-NCL and SKBR-GFP cells using the methylene blue viability assay (means ±SD). E. Cells were pre-treated with GroA as indicated, and total area of colonies formed was determined (means ±SD; *** ( p-value <0.005) - SKBR-GFP compared to SKBR-NCL cells; ^^^ ( p-value <0.005) - comparison between untreated and treated cells of the same cell line).

    Journal: Oncotarget

    Article Title: Nucleolin-binding by ErbB2 enhances tumorigenicity of ErbB2-positive breast cancer

    doi: 10.18632/oncotarget.11323

    Figure Lengend Snippet: A. SKBR3 cells were treated with GroA (10μM), and cell viability was measured by the methylene blue assay at the indicated time points (means ±SD). B. Western-blot analysis of ErbB2 activation, determined by ErbB2 phosphorylation levels, following treatment with GroA (means ±SD). C. SKBR3 cells overexpressing either GFP-nucleolin (SKBR-NCL) or GFP (SKBR-GFP) were treated with GroA (10μM), and cell viability was measured using the methylene blue assay. Left panel , time course curves of cell viability following treatment in SKBR-GFP (control) and SKBR-NCL cells. Right panel , comparison of cell viability in SKBR-GFP and SKBR-NCL cells after 5 days of treatment. Results presented as means ±SD. D. The IC-10, 20 and 50 values for GroA treatment were determined for SKBR-NCL and SKBR-GFP cells using the methylene blue viability assay (means ±SD). E. Cells were pre-treated with GroA as indicated, and total area of colonies formed was determined (means ±SD; *** ( p-value <0.005) - SKBR-GFP compared to SKBR-NCL cells; ^^^ ( p-value <0.005) - comparison between untreated and treated cells of the same cell line).

    Article Snippet: The antibodies used are as follows: monoclonal mouse anti-actin (691001; MP Biomedicals, Santa Ana, CA); polyclonal rabbit anti-ErbB2 (HER2/neu), rabbit anti-nucleolin (C23), rabbit anti-Erk2 and rabbit anti-Akt, monoclonal mouse anti-phosphotyrosine (pY20) and monoclonal mouse anti-GFP and mouse anti-nucleolin (sc-284; sc-13057; sc-154; sc-8312; sc-508; sc-9996; sc-8031, respectively; Santa Cruz Biotechnology, Dallas, TX); monoclonal mouse anti-tubulin (T7816; Sigma-Aldrich); polyclonal rabbit anti-phospho-ErbB2, rabbit anti-phospho-Akt, rabbit anti-phospho-Erk1/2 (2249; 4058; 9101, respectively; Cell Signaling Technology, Danvers, MA); and monoclonal mouse anti-ErbB2 (extracellular; L87), which was a gift from Prof. Y. Yarden, Weizmann Institute of Science, Israel.

    Techniques: Western Blot, Activation Assay, Viability Assay