54653s  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc 54653s
    54653s, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    54653s  (Cell Signaling Technology Inc)


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    Structured Review

    Cell Signaling Technology Inc 54653s
    54653s, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    ar45  (Cell Signaling Technology Inc)


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    Structured Review

    Cell Signaling Technology Inc ar45
    Ar45, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    anti ha polyclonal antibody  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc anti ha polyclonal antibody
    Anti Ha Polyclonal Antibody, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/anti ha polyclonal antibody/product/Cell Signaling Technology Inc
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    ha polyclonal  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc ha polyclonal
    ( a ) HEK293 cells transfected with the indicated plasmids were treated with the indicated inhibitors, lysed, immunoprecipitated with <t>anti-HA</t> antibody and immunoblotted with the indicated antibodies. ( b ) HEK293 cells transfected with the indicated plasmids were treated with increasing concentrations of II-B08, lysed, immunoprecipitated with anti-HA antibody and immunoblotted with the indicated antibodies. ( c ) HEK293 cells transfected with the indicated combination of plasmids and monobodies were lysed, immunoprecipitated with anti-HA antibody and immunoblotted with the indicated antibodies. ( d , e ) HEK293 cells transfected with the indicated plasmids were lysed, immunoprecipitated with anti-HA antibody and immunoblotted with the indicated antibodies. ( f ) Bacterially purified recombinant human GST-H-Ras(WT) was subjected to in vitro kinase assay in the presence of bacterially purified recombinant human active Src kinase. Subsequent dephosphorylation reaction was conducted using increasing amounts of bacterially purified recombinant human active GST-SHP2 followed by immunoprecipitation (IP) with anti-Ras antibody and immunoblotted with the indicated antibodies. The immunoblot data are representative of at least three separate experiments. WCE, whole-cell extract.
    Ha Polyclonal, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/ha polyclonal/product/Cell Signaling Technology Inc
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    1) Product Images from "Inhibition of SHP2-mediated dephosphorylation of Ras suppresses oncogenesis"

    Article Title: Inhibition of SHP2-mediated dephosphorylation of Ras suppresses oncogenesis

    Journal: Nature Communications

    doi: 10.1038/ncomms9859

    ( a ) HEK293 cells transfected with the indicated plasmids were treated with the indicated inhibitors, lysed, immunoprecipitated with anti-HA antibody and immunoblotted with the indicated antibodies. ( b ) HEK293 cells transfected with the indicated plasmids were treated with increasing concentrations of II-B08, lysed, immunoprecipitated with anti-HA antibody and immunoblotted with the indicated antibodies. ( c ) HEK293 cells transfected with the indicated combination of plasmids and monobodies were lysed, immunoprecipitated with anti-HA antibody and immunoblotted with the indicated antibodies. ( d , e ) HEK293 cells transfected with the indicated plasmids were lysed, immunoprecipitated with anti-HA antibody and immunoblotted with the indicated antibodies. ( f ) Bacterially purified recombinant human GST-H-Ras(WT) was subjected to in vitro kinase assay in the presence of bacterially purified recombinant human active Src kinase. Subsequent dephosphorylation reaction was conducted using increasing amounts of bacterially purified recombinant human active GST-SHP2 followed by immunoprecipitation (IP) with anti-Ras antibody and immunoblotted with the indicated antibodies. The immunoblot data are representative of at least three separate experiments. WCE, whole-cell extract.
    Figure Legend Snippet: ( a ) HEK293 cells transfected with the indicated plasmids were treated with the indicated inhibitors, lysed, immunoprecipitated with anti-HA antibody and immunoblotted with the indicated antibodies. ( b ) HEK293 cells transfected with the indicated plasmids were treated with increasing concentrations of II-B08, lysed, immunoprecipitated with anti-HA antibody and immunoblotted with the indicated antibodies. ( c ) HEK293 cells transfected with the indicated combination of plasmids and monobodies were lysed, immunoprecipitated with anti-HA antibody and immunoblotted with the indicated antibodies. ( d , e ) HEK293 cells transfected with the indicated plasmids were lysed, immunoprecipitated with anti-HA antibody and immunoblotted with the indicated antibodies. ( f ) Bacterially purified recombinant human GST-H-Ras(WT) was subjected to in vitro kinase assay in the presence of bacterially purified recombinant human active Src kinase. Subsequent dephosphorylation reaction was conducted using increasing amounts of bacterially purified recombinant human active GST-SHP2 followed by immunoprecipitation (IP) with anti-Ras antibody and immunoblotted with the indicated antibodies. The immunoblot data are representative of at least three separate experiments. WCE, whole-cell extract.

    Techniques Used: Transfection, Immunoprecipitation, Purification, Recombinant, In Vitro, Kinase Assay, De-Phosphorylation Assay, Western Blot

    ( a ) RasB8p0 or p3 astrocytes expressing the HA-H-Ras(12V) transgene derived from GBM-prone mice were either lysed and immunoblotted with the indicated antibodies (top panel) or subjected to SHP2 activity assay (bottom panel). Data represent mean±s.e.m. of three independent experiments performed in triplicate. * P <0.05 Student's t -test compared with RasB8p0. ( b ) RasB8p3 astrocytes were serum starved and pretreated with (+) or without (−) II-B08 followed by treatment with (+) or without (−) 1 ng ml −1 of EGF. Equal amounts of lysates were immunoprecipitated with anti-HA antibody and immunoblotted with the indicated antibodies. ( c ) Normal human astrocytes (NHA), U87 human GBM astrocytes or U87 astrocytes stably expressing EGFRviii deletion mutant (U87-viii) were either lysed and immunoblotted with the indicated antibodies (top panel) or subjected to SHP2 activity assay (bottom panel). Data represent mean±s.e.m. of three independent experiments performed in triplicate. * P <0.05 Student's t -test compared with NHA. ( d ) U87 astrocytes were treated with (+) or without (−) lambda phosphatase (λ PPase), II-B08 or sodium orthovanadate (Na 3 V0 4 ) were lysed, immunoprecipitated with anti-Ras antibody and immunoblotted with indicated antibodies. ( e ) U87 astrocytes were serum starved and pretreated with (+) or without (−) PP2 or II-B08 and treated with (+) or without (−) 1 ng ml −1 of EGF, lysed, immunoprecipitated with anti-Ras antibody and immunoblotted with the indicated antibodies. ( f ) Patient-derived GBM or grade II astrocytoma samples were lysed pulled down using Raf:RBD beads, and immunoblotted with indicated antibodies. The immunoblot data are representative of at least three separate experiments. IP, immunoprecipitation; WCE, whole-cell extract.
    Figure Legend Snippet: ( a ) RasB8p0 or p3 astrocytes expressing the HA-H-Ras(12V) transgene derived from GBM-prone mice were either lysed and immunoblotted with the indicated antibodies (top panel) or subjected to SHP2 activity assay (bottom panel). Data represent mean±s.e.m. of three independent experiments performed in triplicate. * P <0.05 Student's t -test compared with RasB8p0. ( b ) RasB8p3 astrocytes were serum starved and pretreated with (+) or without (−) II-B08 followed by treatment with (+) or without (−) 1 ng ml −1 of EGF. Equal amounts of lysates were immunoprecipitated with anti-HA antibody and immunoblotted with the indicated antibodies. ( c ) Normal human astrocytes (NHA), U87 human GBM astrocytes or U87 astrocytes stably expressing EGFRviii deletion mutant (U87-viii) were either lysed and immunoblotted with the indicated antibodies (top panel) or subjected to SHP2 activity assay (bottom panel). Data represent mean±s.e.m. of three independent experiments performed in triplicate. * P <0.05 Student's t -test compared with NHA. ( d ) U87 astrocytes were treated with (+) or without (−) lambda phosphatase (λ PPase), II-B08 or sodium orthovanadate (Na 3 V0 4 ) were lysed, immunoprecipitated with anti-Ras antibody and immunoblotted with indicated antibodies. ( e ) U87 astrocytes were serum starved and pretreated with (+) or without (−) PP2 or II-B08 and treated with (+) or without (−) 1 ng ml −1 of EGF, lysed, immunoprecipitated with anti-Ras antibody and immunoblotted with the indicated antibodies. ( f ) Patient-derived GBM or grade II astrocytoma samples were lysed pulled down using Raf:RBD beads, and immunoblotted with indicated antibodies. The immunoblot data are representative of at least three separate experiments. IP, immunoprecipitation; WCE, whole-cell extract.

    Techniques Used: Expressing, Derivative Assay, Activity Assay, Immunoprecipitation, Stable Transfection, Mutagenesis, Western Blot

    ( a ) HEK293 cells transfected with the indicated plasmids were lysed, immunoprecipitated with anti-Flag antibody and immunoblotted with the indicated antibodies. ( b ) Wild-type MEFs or Src / Yes / Fyn triple-knockout (SYF−/−) MEFs were serum starved and pretreated with (+) or without (−) II-B08 and then treated with (+) or without (−) 1 ng ml −1 of EGF. Equal amounts of lysates were immunoprecipitated with anti-SHP2 antibody and immunoblotted with the indicated antibodies. ( c ) U87 astrocytes were serum starved and pretreated with (+) or without (−) II-B08 followed by a treatment with (+) or without (−) 1 ng ml −1 of EGF, lysed, immunoprecipitated with anti-Ras antibody and immunoblotted with indicated antibodies. ( d ) RasB8p3 astrocytes expressing the HA-H-Ras(12V) transgene were serum starved and pretreated with (+) or without (−) indicated concentration of II-B08 followed by treatment with (+) or without (−) 1 ng ml −1 of EGF, lysed, immunoprecipitated with control rabbit IgG or anti-HA antibody and immunoblotted with the indicated antibodies. The immunoblot data are representative of at least three separate experiments. IP, immunoprecipitation; WCE, whole-cell extract.
    Figure Legend Snippet: ( a ) HEK293 cells transfected with the indicated plasmids were lysed, immunoprecipitated with anti-Flag antibody and immunoblotted with the indicated antibodies. ( b ) Wild-type MEFs or Src / Yes / Fyn triple-knockout (SYF−/−) MEFs were serum starved and pretreated with (+) or without (−) II-B08 and then treated with (+) or without (−) 1 ng ml −1 of EGF. Equal amounts of lysates were immunoprecipitated with anti-SHP2 antibody and immunoblotted with the indicated antibodies. ( c ) U87 astrocytes were serum starved and pretreated with (+) or without (−) II-B08 followed by a treatment with (+) or without (−) 1 ng ml −1 of EGF, lysed, immunoprecipitated with anti-Ras antibody and immunoblotted with indicated antibodies. ( d ) RasB8p3 astrocytes expressing the HA-H-Ras(12V) transgene were serum starved and pretreated with (+) or without (−) indicated concentration of II-B08 followed by treatment with (+) or without (−) 1 ng ml −1 of EGF, lysed, immunoprecipitated with control rabbit IgG or anti-HA antibody and immunoblotted with the indicated antibodies. The immunoblot data are representative of at least three separate experiments. IP, immunoprecipitation; WCE, whole-cell extract.

    Techniques Used: Transfection, Immunoprecipitation, Triple Knockout, Expressing, Concentration Assay, Western Blot

    ( a ) HEK293 cells transfected with the indicated plasmids were lysed, immunoprecipitated with anti-HA antibody or were pulled down using Raf:RBD beads or GTP beads, and immunoblotted with the indicated antibodies. ( b ) HEK293 cells transfected with the indicated plasmids were lysed, immunoprecipitated with anti-Ras antibody and immunoblotted with the indicated antibodies. ( c ) HEK293 cells transfected with the indicated plasmids were lysed, pulled down using Raf:RBD and immunoblotted with the indicated antibodies. ( d ) HEK293 cells transfected with the indicated plasmids were lysed, immunoprecipitated with anti-HA antibody or were pulled down using Raf:RBD beads or GTP beads, and immunoblotted with the indicated antibodies. ( e ) RasB8p3 astrocytes expressing the HA-H-Ras(12V) transgene were serum starved and pretreated with (+) or without (−) II-B08 followed by treatment with (+) or without (−) 1 ng ml −1 of EGF, lysed, pulled down using Raf:RBD beads and immunoblotted with indicated antibodies. ( f ) Equal number of RasB8p3 astrocytes were plated in 96-well plates in sextuplicate and treated with or without (dimethylsulphoxide (DMSO)) increasing concentration of II-B08 for 18 h and alamar blue assay was conducted according to the manufacturer's instructions. Data represent mean±s.e.m. of three independent experiments performed in sextuplicates. * P <0.05 Student's t -test compared with DMSO control. ( g ) Equal number of RasB8 astrocytes were suspended in agar matrix, and treated with or without increasing concentrations of II-B08. Following 10 days incubation, the anchorage-independent growth assay was performed according to the manufacturer's instructions. Data represent mean±s.e.m. of three independent experiments performed in sextuplicates. * P <0.05 Student's t -test compared with DMSO control. Representative images are also shown on the left panel captured using a Nikon Coolpix 995 digital camera mounted on a VWR Vista Vision inverted microscope. Scale bar, 200 μm. The immunoblot data are representative of at least three separate experiments. IP, immunoprecipitation; PD, pull down; WCE, whole-cell extract.
    Figure Legend Snippet: ( a ) HEK293 cells transfected with the indicated plasmids were lysed, immunoprecipitated with anti-HA antibody or were pulled down using Raf:RBD beads or GTP beads, and immunoblotted with the indicated antibodies. ( b ) HEK293 cells transfected with the indicated plasmids were lysed, immunoprecipitated with anti-Ras antibody and immunoblotted with the indicated antibodies. ( c ) HEK293 cells transfected with the indicated plasmids were lysed, pulled down using Raf:RBD and immunoblotted with the indicated antibodies. ( d ) HEK293 cells transfected with the indicated plasmids were lysed, immunoprecipitated with anti-HA antibody or were pulled down using Raf:RBD beads or GTP beads, and immunoblotted with the indicated antibodies. ( e ) RasB8p3 astrocytes expressing the HA-H-Ras(12V) transgene were serum starved and pretreated with (+) or without (−) II-B08 followed by treatment with (+) or without (−) 1 ng ml −1 of EGF, lysed, pulled down using Raf:RBD beads and immunoblotted with indicated antibodies. ( f ) Equal number of RasB8p3 astrocytes were plated in 96-well plates in sextuplicate and treated with or without (dimethylsulphoxide (DMSO)) increasing concentration of II-B08 for 18 h and alamar blue assay was conducted according to the manufacturer's instructions. Data represent mean±s.e.m. of three independent experiments performed in sextuplicates. * P <0.05 Student's t -test compared with DMSO control. ( g ) Equal number of RasB8 astrocytes were suspended in agar matrix, and treated with or without increasing concentrations of II-B08. Following 10 days incubation, the anchorage-independent growth assay was performed according to the manufacturer's instructions. Data represent mean±s.e.m. of three independent experiments performed in sextuplicates. * P <0.05 Student's t -test compared with DMSO control. Representative images are also shown on the left panel captured using a Nikon Coolpix 995 digital camera mounted on a VWR Vista Vision inverted microscope. Scale bar, 200 μm. The immunoblot data are representative of at least three separate experiments. IP, immunoprecipitation; PD, pull down; WCE, whole-cell extract.

    Techniques Used: Transfection, Immunoprecipitation, Expressing, Concentration Assay, Alamar Blue Assay, Incubation, Growth Assay, Inverted Microscopy, Western Blot

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    Cell Signaling Technology Inc 54653s
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    Cell Signaling Technology Inc anti ha polyclonal antibody
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    Cell Signaling Technology Inc ha polyclonal
    ( a ) HEK293 cells transfected with the indicated plasmids were treated with the indicated inhibitors, lysed, immunoprecipitated with <t>anti-HA</t> antibody and immunoblotted with the indicated antibodies. ( b ) HEK293 cells transfected with the indicated plasmids were treated with increasing concentrations of II-B08, lysed, immunoprecipitated with anti-HA antibody and immunoblotted with the indicated antibodies. ( c ) HEK293 cells transfected with the indicated combination of plasmids and monobodies were lysed, immunoprecipitated with anti-HA antibody and immunoblotted with the indicated antibodies. ( d , e ) HEK293 cells transfected with the indicated plasmids were lysed, immunoprecipitated with anti-HA antibody and immunoblotted with the indicated antibodies. ( f ) Bacterially purified recombinant human GST-H-Ras(WT) was subjected to in vitro kinase assay in the presence of bacterially purified recombinant human active Src kinase. Subsequent dephosphorylation reaction was conducted using increasing amounts of bacterially purified recombinant human active GST-SHP2 followed by immunoprecipitation (IP) with anti-Ras antibody and immunoblotted with the indicated antibodies. The immunoblot data are representative of at least three separate experiments. WCE, whole-cell extract.
    Ha Polyclonal, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/ha polyclonal/product/Cell Signaling Technology Inc
    Average 93 stars, based on 1 article reviews
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    ( a ) HEK293 cells transfected with the indicated plasmids were treated with the indicated inhibitors, lysed, immunoprecipitated with anti-HA antibody and immunoblotted with the indicated antibodies. ( b ) HEK293 cells transfected with the indicated plasmids were treated with increasing concentrations of II-B08, lysed, immunoprecipitated with anti-HA antibody and immunoblotted with the indicated antibodies. ( c ) HEK293 cells transfected with the indicated combination of plasmids and monobodies were lysed, immunoprecipitated with anti-HA antibody and immunoblotted with the indicated antibodies. ( d , e ) HEK293 cells transfected with the indicated plasmids were lysed, immunoprecipitated with anti-HA antibody and immunoblotted with the indicated antibodies. ( f ) Bacterially purified recombinant human GST-H-Ras(WT) was subjected to in vitro kinase assay in the presence of bacterially purified recombinant human active Src kinase. Subsequent dephosphorylation reaction was conducted using increasing amounts of bacterially purified recombinant human active GST-SHP2 followed by immunoprecipitation (IP) with anti-Ras antibody and immunoblotted with the indicated antibodies. The immunoblot data are representative of at least three separate experiments. WCE, whole-cell extract.

    Journal: Nature Communications

    Article Title: Inhibition of SHP2-mediated dephosphorylation of Ras suppresses oncogenesis

    doi: 10.1038/ncomms9859

    Figure Lengend Snippet: ( a ) HEK293 cells transfected with the indicated plasmids were treated with the indicated inhibitors, lysed, immunoprecipitated with anti-HA antibody and immunoblotted with the indicated antibodies. ( b ) HEK293 cells transfected with the indicated plasmids were treated with increasing concentrations of II-B08, lysed, immunoprecipitated with anti-HA antibody and immunoblotted with the indicated antibodies. ( c ) HEK293 cells transfected with the indicated combination of plasmids and monobodies were lysed, immunoprecipitated with anti-HA antibody and immunoblotted with the indicated antibodies. ( d , e ) HEK293 cells transfected with the indicated plasmids were lysed, immunoprecipitated with anti-HA antibody and immunoblotted with the indicated antibodies. ( f ) Bacterially purified recombinant human GST-H-Ras(WT) was subjected to in vitro kinase assay in the presence of bacterially purified recombinant human active Src kinase. Subsequent dephosphorylation reaction was conducted using increasing amounts of bacterially purified recombinant human active GST-SHP2 followed by immunoprecipitation (IP) with anti-Ras antibody and immunoblotted with the indicated antibodies. The immunoblot data are representative of at least three separate experiments. WCE, whole-cell extract.

    Article Snippet: Rabbit polyclonal antibodies against Src, phosphorylated (p)Src(Y416), pAKT, AKT, glutathione S -transferase (GST), pSHP2(Y542), SHP2 and HA (polyclonal) were obtained from Cell Signaling Technologies.

    Techniques: Transfection, Immunoprecipitation, Purification, Recombinant, In Vitro, Kinase Assay, De-Phosphorylation Assay, Western Blot

    ( a ) RasB8p0 or p3 astrocytes expressing the HA-H-Ras(12V) transgene derived from GBM-prone mice were either lysed and immunoblotted with the indicated antibodies (top panel) or subjected to SHP2 activity assay (bottom panel). Data represent mean±s.e.m. of three independent experiments performed in triplicate. * P <0.05 Student's t -test compared with RasB8p0. ( b ) RasB8p3 astrocytes were serum starved and pretreated with (+) or without (−) II-B08 followed by treatment with (+) or without (−) 1 ng ml −1 of EGF. Equal amounts of lysates were immunoprecipitated with anti-HA antibody and immunoblotted with the indicated antibodies. ( c ) Normal human astrocytes (NHA), U87 human GBM astrocytes or U87 astrocytes stably expressing EGFRviii deletion mutant (U87-viii) were either lysed and immunoblotted with the indicated antibodies (top panel) or subjected to SHP2 activity assay (bottom panel). Data represent mean±s.e.m. of three independent experiments performed in triplicate. * P <0.05 Student's t -test compared with NHA. ( d ) U87 astrocytes were treated with (+) or without (−) lambda phosphatase (λ PPase), II-B08 or sodium orthovanadate (Na 3 V0 4 ) were lysed, immunoprecipitated with anti-Ras antibody and immunoblotted with indicated antibodies. ( e ) U87 astrocytes were serum starved and pretreated with (+) or without (−) PP2 or II-B08 and treated with (+) or without (−) 1 ng ml −1 of EGF, lysed, immunoprecipitated with anti-Ras antibody and immunoblotted with the indicated antibodies. ( f ) Patient-derived GBM or grade II astrocytoma samples were lysed pulled down using Raf:RBD beads, and immunoblotted with indicated antibodies. The immunoblot data are representative of at least three separate experiments. IP, immunoprecipitation; WCE, whole-cell extract.

    Journal: Nature Communications

    Article Title: Inhibition of SHP2-mediated dephosphorylation of Ras suppresses oncogenesis

    doi: 10.1038/ncomms9859

    Figure Lengend Snippet: ( a ) RasB8p0 or p3 astrocytes expressing the HA-H-Ras(12V) transgene derived from GBM-prone mice were either lysed and immunoblotted with the indicated antibodies (top panel) or subjected to SHP2 activity assay (bottom panel). Data represent mean±s.e.m. of three independent experiments performed in triplicate. * P <0.05 Student's t -test compared with RasB8p0. ( b ) RasB8p3 astrocytes were serum starved and pretreated with (+) or without (−) II-B08 followed by treatment with (+) or without (−) 1 ng ml −1 of EGF. Equal amounts of lysates were immunoprecipitated with anti-HA antibody and immunoblotted with the indicated antibodies. ( c ) Normal human astrocytes (NHA), U87 human GBM astrocytes or U87 astrocytes stably expressing EGFRviii deletion mutant (U87-viii) were either lysed and immunoblotted with the indicated antibodies (top panel) or subjected to SHP2 activity assay (bottom panel). Data represent mean±s.e.m. of three independent experiments performed in triplicate. * P <0.05 Student's t -test compared with NHA. ( d ) U87 astrocytes were treated with (+) or without (−) lambda phosphatase (λ PPase), II-B08 or sodium orthovanadate (Na 3 V0 4 ) were lysed, immunoprecipitated with anti-Ras antibody and immunoblotted with indicated antibodies. ( e ) U87 astrocytes were serum starved and pretreated with (+) or without (−) PP2 or II-B08 and treated with (+) or without (−) 1 ng ml −1 of EGF, lysed, immunoprecipitated with anti-Ras antibody and immunoblotted with the indicated antibodies. ( f ) Patient-derived GBM or grade II astrocytoma samples were lysed pulled down using Raf:RBD beads, and immunoblotted with indicated antibodies. The immunoblot data are representative of at least three separate experiments. IP, immunoprecipitation; WCE, whole-cell extract.

    Article Snippet: Rabbit polyclonal antibodies against Src, phosphorylated (p)Src(Y416), pAKT, AKT, glutathione S -transferase (GST), pSHP2(Y542), SHP2 and HA (polyclonal) were obtained from Cell Signaling Technologies.

    Techniques: Expressing, Derivative Assay, Activity Assay, Immunoprecipitation, Stable Transfection, Mutagenesis, Western Blot

    ( a ) HEK293 cells transfected with the indicated plasmids were lysed, immunoprecipitated with anti-Flag antibody and immunoblotted with the indicated antibodies. ( b ) Wild-type MEFs or Src / Yes / Fyn triple-knockout (SYF−/−) MEFs were serum starved and pretreated with (+) or without (−) II-B08 and then treated with (+) or without (−) 1 ng ml −1 of EGF. Equal amounts of lysates were immunoprecipitated with anti-SHP2 antibody and immunoblotted with the indicated antibodies. ( c ) U87 astrocytes were serum starved and pretreated with (+) or without (−) II-B08 followed by a treatment with (+) or without (−) 1 ng ml −1 of EGF, lysed, immunoprecipitated with anti-Ras antibody and immunoblotted with indicated antibodies. ( d ) RasB8p3 astrocytes expressing the HA-H-Ras(12V) transgene were serum starved and pretreated with (+) or without (−) indicated concentration of II-B08 followed by treatment with (+) or without (−) 1 ng ml −1 of EGF, lysed, immunoprecipitated with control rabbit IgG or anti-HA antibody and immunoblotted with the indicated antibodies. The immunoblot data are representative of at least three separate experiments. IP, immunoprecipitation; WCE, whole-cell extract.

    Journal: Nature Communications

    Article Title: Inhibition of SHP2-mediated dephosphorylation of Ras suppresses oncogenesis

    doi: 10.1038/ncomms9859

    Figure Lengend Snippet: ( a ) HEK293 cells transfected with the indicated plasmids were lysed, immunoprecipitated with anti-Flag antibody and immunoblotted with the indicated antibodies. ( b ) Wild-type MEFs or Src / Yes / Fyn triple-knockout (SYF−/−) MEFs were serum starved and pretreated with (+) or without (−) II-B08 and then treated with (+) or without (−) 1 ng ml −1 of EGF. Equal amounts of lysates were immunoprecipitated with anti-SHP2 antibody and immunoblotted with the indicated antibodies. ( c ) U87 astrocytes were serum starved and pretreated with (+) or without (−) II-B08 followed by a treatment with (+) or without (−) 1 ng ml −1 of EGF, lysed, immunoprecipitated with anti-Ras antibody and immunoblotted with indicated antibodies. ( d ) RasB8p3 astrocytes expressing the HA-H-Ras(12V) transgene were serum starved and pretreated with (+) or without (−) indicated concentration of II-B08 followed by treatment with (+) or without (−) 1 ng ml −1 of EGF, lysed, immunoprecipitated with control rabbit IgG or anti-HA antibody and immunoblotted with the indicated antibodies. The immunoblot data are representative of at least three separate experiments. IP, immunoprecipitation; WCE, whole-cell extract.

    Article Snippet: Rabbit polyclonal antibodies against Src, phosphorylated (p)Src(Y416), pAKT, AKT, glutathione S -transferase (GST), pSHP2(Y542), SHP2 and HA (polyclonal) were obtained from Cell Signaling Technologies.

    Techniques: Transfection, Immunoprecipitation, Triple Knockout, Expressing, Concentration Assay, Western Blot

    ( a ) HEK293 cells transfected with the indicated plasmids were lysed, immunoprecipitated with anti-HA antibody or were pulled down using Raf:RBD beads or GTP beads, and immunoblotted with the indicated antibodies. ( b ) HEK293 cells transfected with the indicated plasmids were lysed, immunoprecipitated with anti-Ras antibody and immunoblotted with the indicated antibodies. ( c ) HEK293 cells transfected with the indicated plasmids were lysed, pulled down using Raf:RBD and immunoblotted with the indicated antibodies. ( d ) HEK293 cells transfected with the indicated plasmids were lysed, immunoprecipitated with anti-HA antibody or were pulled down using Raf:RBD beads or GTP beads, and immunoblotted with the indicated antibodies. ( e ) RasB8p3 astrocytes expressing the HA-H-Ras(12V) transgene were serum starved and pretreated with (+) or without (−) II-B08 followed by treatment with (+) or without (−) 1 ng ml −1 of EGF, lysed, pulled down using Raf:RBD beads and immunoblotted with indicated antibodies. ( f ) Equal number of RasB8p3 astrocytes were plated in 96-well plates in sextuplicate and treated with or without (dimethylsulphoxide (DMSO)) increasing concentration of II-B08 for 18 h and alamar blue assay was conducted according to the manufacturer's instructions. Data represent mean±s.e.m. of three independent experiments performed in sextuplicates. * P <0.05 Student's t -test compared with DMSO control. ( g ) Equal number of RasB8 astrocytes were suspended in agar matrix, and treated with or without increasing concentrations of II-B08. Following 10 days incubation, the anchorage-independent growth assay was performed according to the manufacturer's instructions. Data represent mean±s.e.m. of three independent experiments performed in sextuplicates. * P <0.05 Student's t -test compared with DMSO control. Representative images are also shown on the left panel captured using a Nikon Coolpix 995 digital camera mounted on a VWR Vista Vision inverted microscope. Scale bar, 200 μm. The immunoblot data are representative of at least three separate experiments. IP, immunoprecipitation; PD, pull down; WCE, whole-cell extract.

    Journal: Nature Communications

    Article Title: Inhibition of SHP2-mediated dephosphorylation of Ras suppresses oncogenesis

    doi: 10.1038/ncomms9859

    Figure Lengend Snippet: ( a ) HEK293 cells transfected with the indicated plasmids were lysed, immunoprecipitated with anti-HA antibody or were pulled down using Raf:RBD beads or GTP beads, and immunoblotted with the indicated antibodies. ( b ) HEK293 cells transfected with the indicated plasmids were lysed, immunoprecipitated with anti-Ras antibody and immunoblotted with the indicated antibodies. ( c ) HEK293 cells transfected with the indicated plasmids were lysed, pulled down using Raf:RBD and immunoblotted with the indicated antibodies. ( d ) HEK293 cells transfected with the indicated plasmids were lysed, immunoprecipitated with anti-HA antibody or were pulled down using Raf:RBD beads or GTP beads, and immunoblotted with the indicated antibodies. ( e ) RasB8p3 astrocytes expressing the HA-H-Ras(12V) transgene were serum starved and pretreated with (+) or without (−) II-B08 followed by treatment with (+) or without (−) 1 ng ml −1 of EGF, lysed, pulled down using Raf:RBD beads and immunoblotted with indicated antibodies. ( f ) Equal number of RasB8p3 astrocytes were plated in 96-well plates in sextuplicate and treated with or without (dimethylsulphoxide (DMSO)) increasing concentration of II-B08 for 18 h and alamar blue assay was conducted according to the manufacturer's instructions. Data represent mean±s.e.m. of three independent experiments performed in sextuplicates. * P <0.05 Student's t -test compared with DMSO control. ( g ) Equal number of RasB8 astrocytes were suspended in agar matrix, and treated with or without increasing concentrations of II-B08. Following 10 days incubation, the anchorage-independent growth assay was performed according to the manufacturer's instructions. Data represent mean±s.e.m. of three independent experiments performed in sextuplicates. * P <0.05 Student's t -test compared with DMSO control. Representative images are also shown on the left panel captured using a Nikon Coolpix 995 digital camera mounted on a VWR Vista Vision inverted microscope. Scale bar, 200 μm. The immunoblot data are representative of at least three separate experiments. IP, immunoprecipitation; PD, pull down; WCE, whole-cell extract.

    Article Snippet: Rabbit polyclonal antibodies against Src, phosphorylated (p)Src(Y416), pAKT, AKT, glutathione S -transferase (GST), pSHP2(Y542), SHP2 and HA (polyclonal) were obtained from Cell Signaling Technologies.

    Techniques: Transfection, Immunoprecipitation, Expressing, Concentration Assay, Alamar Blue Assay, Incubation, Growth Assay, Inverted Microscopy, Western Blot