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Cell Signaling Technology Inc p cdcp1 py734
A. Amino acids are numbered from the first intracellular residue. Phosphorylatable tyrosine residues are shown in bold typeface and are numbered. The ITAM-like motif is shown in a box. B. Alignment of the ITAM-like motif of <t>CDCP1</t> with the consensus sequence of an ITAM motif. Phosphorylatable residues are shown in bold typeface and are numbered according to the corresponding CDCP1 sequence.

A. Amino acids are numbered from the first intracellular residue. Phosphorylatable tyrosine residues are shown in bold typeface and are numbered. The ITAM-like motif is shown in a box. B. Alignment of the ITAM-like motif of <t>CDCP1</t> with the consensus sequence of an ITAM motif. Phosphorylatable residues are shown in bold typeface and are numbered according to the corresponding CDCP1 sequence.

P Cdcp1 Py734, 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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1) Product Images from "The Tyrosine Phosphatase SHP2 Associates with CUB Domain-Containing Protein-1 (CDCP1), Regulating Its Expression at the Cell Surface in a Phosphorylation-Dependent Manner"

Article Title: The Tyrosine Phosphatase SHP2 Associates with CUB Domain-Containing Protein-1 (CDCP1), Regulating Its Expression at the Cell Surface in a Phosphorylation-Dependent Manner

Journal: PLoS ONE

doi: 10.1371/journal.pone.0123472

Figure Legend Snippet: A. Amino acids are numbered from the first intracellular residue. Phosphorylatable tyrosine residues are shown in bold typeface and are numbered. The ITAM-like motif is shown in a box. B. Alignment of the ITAM-like motif of CDCP1 with the consensus sequence of an ITAM motif. Phosphorylatable residues are shown in bold typeface and are numbered according to the corresponding CDCP1 sequence.

Techniques Used: Sequencing

A. PC3 cells were left untreated or were treated for 15 minutes with 25μM pervanadate (PerVO 3 ). The cells were lysed and the lysates were subjected to immunoprecipitation (IP) with anti-CDCP1 control antibodies, as indicated. Total cell lysates and immunoprecipitates were analyzed by western blotting with the antibodies indicated. B and C. HCT 116 cells were transfected with an empty vector or wild-type SHP2 (SHP2-WT-HA) or dominant-negative SHP2 mutant (SHP2-C459S-HA) expression constructs, as indicated. Forty-eight hours after transfection, the cells were left untreated or were treated for 15 minutes with 25μM pervanadate (PerVO 3 ). The cells were lysed and the lysates were subjected to immunoprecipitation (IP) with anti-CDCP1 (A) or anti-HA (B) antibodies, as indicated. Total cell lysates and immunoprecipitates were analyzed by western blotting with the antibodies indicated. The position of the exogenously expressed SHP2 constructs that migrated more slowly, due to a fused HA-Tag, are indicated by an arrowhead. The immunoglobulin heavy chains (IgH) are indicated by asterisks (*). The results shown are representative of at least four independent experiments.

Figure Legend Snippet: A. PC3 cells were left untreated or were treated for 15 minutes with 25μM pervanadate (PerVO 3 ). The cells were lysed and the lysates were subjected to immunoprecipitation (IP) with anti-CDCP1 control antibodies, as indicated. Total cell lysates and immunoprecipitates were analyzed by western blotting with the antibodies indicated. B and C. HCT 116 cells were transfected with an empty vector or wild-type SHP2 (SHP2-WT-HA) or dominant-negative SHP2 mutant (SHP2-C459S-HA) expression constructs, as indicated. Forty-eight hours after transfection, the cells were left untreated or were treated for 15 minutes with 25μM pervanadate (PerVO 3 ). The cells were lysed and the lysates were subjected to immunoprecipitation (IP) with anti-CDCP1 (A) or anti-HA (B) antibodies, as indicated. Total cell lysates and immunoprecipitates were analyzed by western blotting with the antibodies indicated. The position of the exogenously expressed SHP2 constructs that migrated more slowly, due to a fused HA-Tag, are indicated by an arrowhead. The immunoglobulin heavy chains (IgH) are indicated by asterisks (*). The results shown are representative of at least four independent experiments.

Techniques Used: Immunoprecipitation, Western Blot, Transfection, Plasmid Preparation, Dominant Negative Mutation, Mutagenesis, Expressing, Construct

HCT 116 cells were plated at (A) 0.2x10 6 cells/ml (non confluent) or (B) 1.4x10 6 cells/ml (confluent) for 48h. The cells were left untreated or were incubated with 25μM PerVO3 or 5μg/ml anti-CDCP1 antibody, as indicated, for 15 minutes. The cells were lysed and anti-CDCP1 antibodies were used for immunoprecipitation (IP), as indicated and as described in the experimental procedures section. Control immunoprecipitations were performed with irrelevant mouse immunoglobulins (Ig). Total cell lysates and immunoprecipitates were analyzed by western blotting with anti-pTyr, anti-CDCP1 and anti-SHP2 antibodies. The results shown are representative of at least three independent experiments. The position of endogenous SHP2 is indicated by an arrowhead. The asterisks (*) indicate the position of the Ig Heavy chains used for the immunoprecipitation or the triggering of CDCP1.

Figure Legend Snippet: HCT 116 cells were plated at (A) 0.2x10 6 cells/ml (non confluent) or (B) 1.4x10 6 cells/ml (confluent) for 48h. The cells were left untreated or were incubated with 25μM PerVO3 or 5μg/ml anti-CDCP1 antibody, as indicated, for 15 minutes. The cells were lysed and anti-CDCP1 antibodies were used for immunoprecipitation (IP), as indicated and as described in the experimental procedures section. Control immunoprecipitations were performed with irrelevant mouse immunoglobulins (Ig). Total cell lysates and immunoprecipitates were analyzed by western blotting with anti-pTyr, anti-CDCP1 and anti-SHP2 antibodies. The results shown are representative of at least three independent experiments. The position of endogenous SHP2 is indicated by an arrowhead. The asterisks (*) indicate the position of the Ig Heavy chains used for the immunoprecipitation or the triggering of CDCP1.

Techniques Used: Incubation, Immunoprecipitation, Western Blot

HeLa cells stably expressing CDCP1 were left untreated or were treated for 15 minutes with 25μM pervanadate (PerVO 3 ), as indicated. Cell lysates were subjected to GST-pull down assays with 5 μg of GST protein alone (GST only), GST fused to a SHP2 substrate trapping mutant (GST-DACS) or GST fused to the SHP2-SH2 domains (GST-SH2), as mentioned in A and B. The affinity-purified complexes were resolved by SDS-PAGE and analyzed by immunoblotting with the indicated antibodies. In some conditions, particularly in HeLa cells, CDCP1 was detected as two species, the more slowly migrating species being tyrosine-phosphorylated ( , compare lower and middle panels, the arrowhead indicates the slower migrating species). The data shown are representative of more than eight independent experiments.

Figure Legend Snippet: HeLa cells stably expressing CDCP1 were left untreated or were treated for 15 minutes with 25μM pervanadate (PerVO 3 ), as indicated. Cell lysates were subjected to GST-pull down assays with 5 μg of GST protein alone (GST only), GST fused to a SHP2 substrate trapping mutant (GST-DACS) or GST fused to the SHP2-SH2 domains (GST-SH2), as mentioned in A and B. The affinity-purified complexes were resolved by SDS-PAGE and analyzed by immunoblotting with the indicated antibodies. In some conditions, particularly in HeLa cells, CDCP1 was detected as two species, the more slowly migrating species being tyrosine-phosphorylated ( , compare lower and middle panels, the arrowhead indicates the slower migrating species). The data shown are representative of more than eight independent experiments.

Techniques Used: Stable Transfection, Expressing, Mutagenesis, Affinity Purification, SDS Page, Western Blot

A. HeLa cells were transfected with a CDCP1-myc expression construct and were left untreated or were treated, 48h after transfection, with 25μM pervanadate (PerVO 3 ). The cells were lysed and CDCP1 was immunoprecipitated with an anti-Myc antibody (myc). Mouse immunoglobulins were used as a negative control, as indicated. The immune complexes were resolved by SDS-PAGE and transferred on membranes. An overlay assay was performed, as described in the experimental procedures, with a purified SHP2 trapping mutant protein fused to GST (GST-DACS, upper panel). The membrane was then stripped and reprobed with antibodies against phosphorylated tyrosine residues (middle panel) and CDCP1 (bottom panel). In HeLa cells, CDCP1 was detected as a doublet, only the upper band of which (arrowhead) was found to be phosphorylated and to interact with SHP2. B. HeLa cells were transfected with a construct encoding the wild-type CDCP1 (WT), or a CDCP1 protein with substitution of the Y734 or Y743 tyrosine residues (Y734F or Y743F, respectively) or both (DM). All these constructs were Myc-tagged. The cells were treated and lysed as described above. The intensities of the signals were analyzed and were normalized on the signal obtained for the CDCP1 blot (lower panel). The normalized value is considered to be 100% in CDCP1-WT transfected cells treated with PerVO 3 (lane 3). The values are expressed as fold increase or decrease. The data shown are representative of at least five independent experiments.

Figure Legend Snippet: A. HeLa cells were transfected with a CDCP1-myc expression construct and were left untreated or were treated, 48h after transfection, with 25μM pervanadate (PerVO 3 ). The cells were lysed and CDCP1 was immunoprecipitated with an anti-Myc antibody (myc). Mouse immunoglobulins were used as a negative control, as indicated. The immune complexes were resolved by SDS-PAGE and transferred on membranes. An overlay assay was performed, as described in the experimental procedures, with a purified SHP2 trapping mutant protein fused to GST (GST-DACS, upper panel). The membrane was then stripped and reprobed with antibodies against phosphorylated tyrosine residues (middle panel) and CDCP1 (bottom panel). In HeLa cells, CDCP1 was detected as a doublet, only the upper band of which (arrowhead) was found to be phosphorylated and to interact with SHP2. B. HeLa cells were transfected with a construct encoding the wild-type CDCP1 (WT), or a CDCP1 protein with substitution of the Y734 or Y743 tyrosine residues (Y734F or Y743F, respectively) or both (DM). All these constructs were Myc-tagged. The cells were treated and lysed as described above. The intensities of the signals were analyzed and were normalized on the signal obtained for the CDCP1 blot (lower panel). The normalized value is considered to be 100% in CDCP1-WT transfected cells treated with PerVO 3 (lane 3). The values are expressed as fold increase or decrease. The data shown are representative of at least five independent experiments.

Techniques Used: Transfection, Expressing, Construct, Immunoprecipitation, Negative Control, SDS Page, Overlay Assay, Purification, Mutagenesis

A. HeLa cells stably transfected with an empty vector or with a WT-CDCP1 construct were stably transfected with a SHP2-targeting shRNA (D1 or D2), as indicated. Total cell lysates were prepared and analyzed by western blotting with the antibodies indicated. B. Stable HeLa-CDCP1 and HeLa-CDCP1-shSHP2 D1 cell lines (described above and in the experimental procedures) were first incubated with an anti-CDCP1 antibody at 4°C. The cells were washed and incubated at 37°C for the times indicated, to allow internalization of the CDCP1-antibody complexes. The cells were then incubated again at 4°C with the appropriate secondary antibody, and the amount of CDCP1 remaining at the cell surface was analyzed by flow cytometry. The results are indicated as a percentage of membrane CDCP1 ± SEM for three independent experiments. ns: p > 0.05 *: p = 0.03; ****: p = 10 –4 in non-parametric Student's t tests. The data shown are representative of at least three independent experiments performed in triplicate.

Figure Legend Snippet: A. HeLa cells stably transfected with an empty vector or with a WT-CDCP1 construct were stably transfected with a SHP2-targeting shRNA (D1 or D2), as indicated. Total cell lysates were prepared and analyzed by western blotting with the antibodies indicated. B. Stable HeLa-CDCP1 and HeLa-CDCP1-shSHP2 D1 cell lines (described above and in the experimental procedures) were first incubated with an anti-CDCP1 antibody at 4°C. The cells were washed and incubated at 37°C for the times indicated, to allow internalization of the CDCP1-antibody complexes. The cells were then incubated again at 4°C with the appropriate secondary antibody, and the amount of CDCP1 remaining at the cell surface was analyzed by flow cytometry. The results are indicated as a percentage of membrane CDCP1 ± SEM for three independent experiments. ns: p > 0.05 *: p = 0.03; ****: p = 10 –4 in non-parametric Student's t tests. The data shown are representative of at least three independent experiments performed in triplicate.

Techniques Used: Stable Transfection, Transfection, Plasmid Preparation, Construct, shRNA, Western Blot, Incubation, Flow Cytometry

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Journal: PLoS ONE

Article Title: The Tyrosine Phosphatase SHP2 Associates with CUB Domain-Containing Protein-1 (CDCP1), Regulating Its Expression at the Cell Surface in a Phosphorylation-Dependent Manner

doi: 10.1371/journal.pone.0123472

Figure Lengend Snippet: A. Amino acids are numbered from the first intracellular residue. Phosphorylatable tyrosine residues are shown in bold typeface and are numbered. The ITAM-like motif is shown in a box. B. Alignment of the ITAM-like motif of CDCP1 with the consensus sequence of an ITAM motif. Phosphorylatable residues are shown in bold typeface and are numbered according to the corresponding CDCP1 sequence.

Article Snippet: Polyclonal antibodies directed against p-CDCP1 (pY734) were purchased from Cell Signaling Technology (Ozyme, Saint-Quentin-en-Yvelines, France).

Techniques: Sequencing

Journal: PLoS ONE

Article Title: The Tyrosine Phosphatase SHP2 Associates with CUB Domain-Containing Protein-1 (CDCP1), Regulating Its Expression at the Cell Surface in a Phosphorylation-Dependent Manner

doi: 10.1371/journal.pone.0123472

Figure Lengend Snippet: A. PC3 cells were left untreated or were treated for 15 minutes with 25μM pervanadate (PerVO 3 ). The cells were lysed and the lysates were subjected to immunoprecipitation (IP) with anti-CDCP1 control antibodies, as indicated. Total cell lysates and immunoprecipitates were analyzed by western blotting with the antibodies indicated. B and C. HCT 116 cells were transfected with an empty vector or wild-type SHP2 (SHP2-WT-HA) or dominant-negative SHP2 mutant (SHP2-C459S-HA) expression constructs, as indicated. Forty-eight hours after transfection, the cells were left untreated or were treated for 15 minutes with 25μM pervanadate (PerVO 3 ). The cells were lysed and the lysates were subjected to immunoprecipitation (IP) with anti-CDCP1 (A) or anti-HA (B) antibodies, as indicated. Total cell lysates and immunoprecipitates were analyzed by western blotting with the antibodies indicated. The position of the exogenously expressed SHP2 constructs that migrated more slowly, due to a fused HA-Tag, are indicated by an arrowhead. The immunoglobulin heavy chains (IgH) are indicated by asterisks (*). The results shown are representative of at least four independent experiments.

Article Snippet: Polyclonal antibodies directed against p-CDCP1 (pY734) were purchased from Cell Signaling Technology (Ozyme, Saint-Quentin-en-Yvelines, France).

Techniques: Immunoprecipitation, Western Blot, Transfection, Plasmid Preparation, Dominant Negative Mutation, Mutagenesis, Expressing, Construct

Journal: PLoS ONE

Article Title: The Tyrosine Phosphatase SHP2 Associates with CUB Domain-Containing Protein-1 (CDCP1), Regulating Its Expression at the Cell Surface in a Phosphorylation-Dependent Manner

doi: 10.1371/journal.pone.0123472

Figure Lengend Snippet: HCT 116 cells were plated at (A) 0.2x10 6 cells/ml (non confluent) or (B) 1.4x10 6 cells/ml (confluent) for 48h. The cells were left untreated or were incubated with 25μM PerVO3 or 5μg/ml anti-CDCP1 antibody, as indicated, for 15 minutes. The cells were lysed and anti-CDCP1 antibodies were used for immunoprecipitation (IP), as indicated and as described in the experimental procedures section. Control immunoprecipitations were performed with irrelevant mouse immunoglobulins (Ig). Total cell lysates and immunoprecipitates were analyzed by western blotting with anti-pTyr, anti-CDCP1 and anti-SHP2 antibodies. The results shown are representative of at least three independent experiments. The position of endogenous SHP2 is indicated by an arrowhead. The asterisks (*) indicate the position of the Ig Heavy chains used for the immunoprecipitation or the triggering of CDCP1.

Article Snippet: Polyclonal antibodies directed against p-CDCP1 (pY734) were purchased from Cell Signaling Technology (Ozyme, Saint-Quentin-en-Yvelines, France).

Techniques: Incubation, Immunoprecipitation, Western Blot

Journal: PLoS ONE

Article Title: The Tyrosine Phosphatase SHP2 Associates with CUB Domain-Containing Protein-1 (CDCP1), Regulating Its Expression at the Cell Surface in a Phosphorylation-Dependent Manner

doi: 10.1371/journal.pone.0123472

Figure Lengend Snippet: HeLa cells stably expressing CDCP1 were left untreated or were treated for 15 minutes with 25μM pervanadate (PerVO 3 ), as indicated. Cell lysates were subjected to GST-pull down assays with 5 μg of GST protein alone (GST only), GST fused to a SHP2 substrate trapping mutant (GST-DACS) or GST fused to the SHP2-SH2 domains (GST-SH2), as mentioned in A and B. The affinity-purified complexes were resolved by SDS-PAGE and analyzed by immunoblotting with the indicated antibodies. In some conditions, particularly in HeLa cells, CDCP1 was detected as two species, the more slowly migrating species being tyrosine-phosphorylated ( , compare lower and middle panels, the arrowhead indicates the slower migrating species). The data shown are representative of more than eight independent experiments.

Article Snippet: Polyclonal antibodies directed against p-CDCP1 (pY734) were purchased from Cell Signaling Technology (Ozyme, Saint-Quentin-en-Yvelines, France).

Techniques: Stable Transfection, Expressing, Mutagenesis, Affinity Purification, SDS Page, Western Blot

Journal: PLoS ONE

Article Title: The Tyrosine Phosphatase SHP2 Associates with CUB Domain-Containing Protein-1 (CDCP1), Regulating Its Expression at the Cell Surface in a Phosphorylation-Dependent Manner

doi: 10.1371/journal.pone.0123472

Figure Lengend Snippet: A. HeLa cells were transfected with a CDCP1-myc expression construct and were left untreated or were treated, 48h after transfection, with 25μM pervanadate (PerVO 3 ). The cells were lysed and CDCP1 was immunoprecipitated with an anti-Myc antibody (myc). Mouse immunoglobulins were used as a negative control, as indicated. The immune complexes were resolved by SDS-PAGE and transferred on membranes. An overlay assay was performed, as described in the experimental procedures, with a purified SHP2 trapping mutant protein fused to GST (GST-DACS, upper panel). The membrane was then stripped and reprobed with antibodies against phosphorylated tyrosine residues (middle panel) and CDCP1 (bottom panel). In HeLa cells, CDCP1 was detected as a doublet, only the upper band of which (arrowhead) was found to be phosphorylated and to interact with SHP2. B. HeLa cells were transfected with a construct encoding the wild-type CDCP1 (WT), or a CDCP1 protein with substitution of the Y734 or Y743 tyrosine residues (Y734F or Y743F, respectively) or both (DM). All these constructs were Myc-tagged. The cells were treated and lysed as described above. The intensities of the signals were analyzed and were normalized on the signal obtained for the CDCP1 blot (lower panel). The normalized value is considered to be 100% in CDCP1-WT transfected cells treated with PerVO 3 (lane 3). The values are expressed as fold increase or decrease. The data shown are representative of at least five independent experiments.

Article Snippet: Polyclonal antibodies directed against p-CDCP1 (pY734) were purchased from Cell Signaling Technology (Ozyme, Saint-Quentin-en-Yvelines, France).

Techniques: Transfection, Expressing, Construct, Immunoprecipitation, Negative Control, SDS Page, Overlay Assay, Purification, Mutagenesis

Journal: PLoS ONE

Article Title: The Tyrosine Phosphatase SHP2 Associates with CUB Domain-Containing Protein-1 (CDCP1), Regulating Its Expression at the Cell Surface in a Phosphorylation-Dependent Manner

doi: 10.1371/journal.pone.0123472

Figure Lengend Snippet: A. HeLa cells stably transfected with an empty vector or with a WT-CDCP1 construct were stably transfected with a SHP2-targeting shRNA (D1 or D2), as indicated. Total cell lysates were prepared and analyzed by western blotting with the antibodies indicated. B. Stable HeLa-CDCP1 and HeLa-CDCP1-shSHP2 D1 cell lines (described above and in the experimental procedures) were first incubated with an anti-CDCP1 antibody at 4°C. The cells were washed and incubated at 37°C for the times indicated, to allow internalization of the CDCP1-antibody complexes. The cells were then incubated again at 4°C with the appropriate secondary antibody, and the amount of CDCP1 remaining at the cell surface was analyzed by flow cytometry. The results are indicated as a percentage of membrane CDCP1 ± SEM for three independent experiments. ns: p > 0.05 *: p = 0.03; ****: p = 10 –4 in non-parametric Student's t tests. The data shown are representative of at least three independent experiments performed in triplicate.

Article Snippet: Polyclonal antibodies directed against p-CDCP1 (pY734) were purchased from Cell Signaling Technology (Ozyme, Saint-Quentin-en-Yvelines, France).

Techniques: Stable Transfection, Transfection, Plasmid Preparation, Construct, shRNA, Western Blot, Incubation, Flow Cytometry