anti cdcp1 py734  (Cell Signaling Technology Inc)


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    Structured Review

    Cell Signaling Technology Inc anti cdcp1 py734
    (A, B, C, D) MDCK cysts embedded within the collagen matrix were pretreated with NK4 (1 μg/ml), Torin1 (+, 50 nM; ++, 100 nM), rapamycin (+ Rapa, 50 nM; ++ Rapa, 100 nM), dasatinib (20 nM, Dasa), saracatinib (10 μM, Sara), or simvastatin (2 μM, Statin) and then incubated in the presence of HGF (50 ng/ml) for 1 d. (A) Ki67 was visualized with an Alexa Fluor 488–conjugated antibody (green), and actin filaments were stained with Alexa Fluor 594–phalloidin (magenta); arrowheads indicate transiently formed protrusions. Diameter (μm) of cysts (n = 100). (B) Dotted blue line indicates the average diameter of non-treated cysts. (C) Ratio of Ki67-positive cell in cyst (n = 50). (D) Fraction of the total number of cysts counted (n > 100) with protrusions. (E) MDCK cysts embedded within the collagen matrix were incubated in the presence of HGF (50 ng/ml) for the indicated time periods. Activated Src (pY418) was visualized with an Alexa Fluor 488–conjugated antibody (green), and actin filaments were stained with Alexa Fluor 594-phalloidin (magenta); arrowheads indicate transiently formed protrusions. (F) mCherry-GPI–overexpressing MDCK cysts embedded within the collagen matrix were incubated in the presence of HGF (50 ng/ml) for the indicated time periods. Actin filaments were stained with Alexa Fluor 488-phalloidin (green); arrowheads indicate transiently formed protrusions. (G) MDCK cysts embedded within the collagen matrix were incubated in the presence of HGF (50 ng/ml) for the indicated time periods. Localization of <t>CDCP1</t> was visualized using an Alexa Fluor 488–conjugated antibody (green), and actin filaments were stained with Alexa Fluor 594-phalloidin (magenta); arrowheads indicate transiently formed protrusions. (H, I, J) Wild-type and CDCP1 -knockout MDCK cysts were incubated in the presence of HGF (50 ng/ml) for 1 d. (H) Ki67 was visualized with an Alexa Fluor 488-conjugated antibody (green), and actin filaments were stained with Alexa Fluor 594-phalloidin (magenta); arrowheads indicate transiently formed protrusions. (I) Diameter (μm) of cysts (n = 100). (J) Fraction of the total number of cysts counted (n > 100) with protrusions. Scale bars: 10 μm. Data information: In (B, C, D, I, J), the mean ratios ± SD were obtained from three independent experiments. * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001; NS, not significantly different; two-way ANOVA was calculated compared with HGF-treated cysts.
    Anti Cdcp1 Py734, 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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    Images

    1) Product Images from "CDCP1 promotes compensatory renal growth by integrating Src and Met signaling"

    Article Title: CDCP1 promotes compensatory renal growth by integrating Src and Met signaling

    Journal: Life Science Alliance

    doi: 10.26508/lsa.202000832

    (A, B, C, D) MDCK cysts embedded within the collagen matrix were pretreated with NK4 (1 μg/ml), Torin1 (+, 50 nM; ++, 100 nM), rapamycin (+ Rapa, 50 nM; ++ Rapa, 100 nM), dasatinib (20 nM, Dasa), saracatinib (10 μM, Sara), or simvastatin (2 μM, Statin) and then incubated in the presence of HGF (50 ng/ml) for 1 d. (A) Ki67 was visualized with an Alexa Fluor 488–conjugated antibody (green), and actin filaments were stained with Alexa Fluor 594–phalloidin (magenta); arrowheads indicate transiently formed protrusions. Diameter (μm) of cysts (n = 100). (B) Dotted blue line indicates the average diameter of non-treated cysts. (C) Ratio of Ki67-positive cell in cyst (n = 50). (D) Fraction of the total number of cysts counted (n > 100) with protrusions. (E) MDCK cysts embedded within the collagen matrix were incubated in the presence of HGF (50 ng/ml) for the indicated time periods. Activated Src (pY418) was visualized with an Alexa Fluor 488–conjugated antibody (green), and actin filaments were stained with Alexa Fluor 594-phalloidin (magenta); arrowheads indicate transiently formed protrusions. (F) mCherry-GPI–overexpressing MDCK cysts embedded within the collagen matrix were incubated in the presence of HGF (50 ng/ml) for the indicated time periods. Actin filaments were stained with Alexa Fluor 488-phalloidin (green); arrowheads indicate transiently formed protrusions. (G) MDCK cysts embedded within the collagen matrix were incubated in the presence of HGF (50 ng/ml) for the indicated time periods. Localization of CDCP1 was visualized using an Alexa Fluor 488–conjugated antibody (green), and actin filaments were stained with Alexa Fluor 594-phalloidin (magenta); arrowheads indicate transiently formed protrusions. (H, I, J) Wild-type and CDCP1 -knockout MDCK cysts were incubated in the presence of HGF (50 ng/ml) for 1 d. (H) Ki67 was visualized with an Alexa Fluor 488-conjugated antibody (green), and actin filaments were stained with Alexa Fluor 594-phalloidin (magenta); arrowheads indicate transiently formed protrusions. (I) Diameter (μm) of cysts (n = 100). (J) Fraction of the total number of cysts counted (n > 100) with protrusions. Scale bars: 10 μm. Data information: In (B, C, D, I, J), the mean ratios ± SD were obtained from three independent experiments. * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001; NS, not significantly different; two-way ANOVA was calculated compared with HGF-treated cysts.
    Figure Legend Snippet: (A, B, C, D) MDCK cysts embedded within the collagen matrix were pretreated with NK4 (1 μg/ml), Torin1 (+, 50 nM; ++, 100 nM), rapamycin (+ Rapa, 50 nM; ++ Rapa, 100 nM), dasatinib (20 nM, Dasa), saracatinib (10 μM, Sara), or simvastatin (2 μM, Statin) and then incubated in the presence of HGF (50 ng/ml) for 1 d. (A) Ki67 was visualized with an Alexa Fluor 488–conjugated antibody (green), and actin filaments were stained with Alexa Fluor 594–phalloidin (magenta); arrowheads indicate transiently formed protrusions. Diameter (μm) of cysts (n = 100). (B) Dotted blue line indicates the average diameter of non-treated cysts. (C) Ratio of Ki67-positive cell in cyst (n = 50). (D) Fraction of the total number of cysts counted (n > 100) with protrusions. (E) MDCK cysts embedded within the collagen matrix were incubated in the presence of HGF (50 ng/ml) for the indicated time periods. Activated Src (pY418) was visualized with an Alexa Fluor 488–conjugated antibody (green), and actin filaments were stained with Alexa Fluor 594-phalloidin (magenta); arrowheads indicate transiently formed protrusions. (F) mCherry-GPI–overexpressing MDCK cysts embedded within the collagen matrix were incubated in the presence of HGF (50 ng/ml) for the indicated time periods. Actin filaments were stained with Alexa Fluor 488-phalloidin (green); arrowheads indicate transiently formed protrusions. (G) MDCK cysts embedded within the collagen matrix were incubated in the presence of HGF (50 ng/ml) for the indicated time periods. Localization of CDCP1 was visualized using an Alexa Fluor 488–conjugated antibody (green), and actin filaments were stained with Alexa Fluor 594-phalloidin (magenta); arrowheads indicate transiently formed protrusions. (H, I, J) Wild-type and CDCP1 -knockout MDCK cysts were incubated in the presence of HGF (50 ng/ml) for 1 d. (H) Ki67 was visualized with an Alexa Fluor 488-conjugated antibody (green), and actin filaments were stained with Alexa Fluor 594-phalloidin (magenta); arrowheads indicate transiently formed protrusions. (I) Diameter (μm) of cysts (n = 100). (J) Fraction of the total number of cysts counted (n > 100) with protrusions. Scale bars: 10 μm. Data information: In (B, C, D, I, J), the mean ratios ± SD were obtained from three independent experiments. * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001; NS, not significantly different; two-way ANOVA was calculated compared with HGF-treated cysts.

    Techniques Used: Incubation, Staining, Knock-Out

    (A) Schematic structure of CDCP1. In the cytoplasmic region, CDCP1 contains a Src association motif around Tyr734 and two palmitoylation sites (Cys residues 689 and 690), which are required for the localization of CDCP1 in lipid rafts. Upon phosphorylation at Tyr734 by Src, CDCP1 is additionally phosphorylated at Tyr762, resulting in direct association with PKCδ, which promotes cell migration. In the extracellular region, CDCP1 contains three CUB domains that are required for protein–protein interactions. CDCP1 also harbors a proteolytic cleavage (shedding) site between the first and second CUB domains and can, thus, be present in either the full-length or cleaved form depending on the cellular context. TM, transmembrane domain. (B) Schematic diagram of CRISPR/Cas9-based generation of CDCP1 -knockout MDCK cells. Blue indicates the PAM sequence and bold letters indicate the start codon. The red arrowhead indicates the cleavage site. (C) Immunoblotting analysis of CDCP1 -knockout MDCK cells. Lysates from wild-type and CDCP1 -knockout MDCK cells were subjected to immunoblotting using the indicated antibodies. (D) CDCP1 -knockout MDCK cysts were incubated in the presence of hepatocyte growth factor (50 ng/ml) for 4 and 6 d. Ki67 was visualized with an Alexa Fluor 488–conjugated antibody (green) and actin filaments were stained with Alexa Fluor 594-phalloidin (magenta). Scale bars: 10 μm. Source data are available for this figure.
    Figure Legend Snippet: (A) Schematic structure of CDCP1. In the cytoplasmic region, CDCP1 contains a Src association motif around Tyr734 and two palmitoylation sites (Cys residues 689 and 690), which are required for the localization of CDCP1 in lipid rafts. Upon phosphorylation at Tyr734 by Src, CDCP1 is additionally phosphorylated at Tyr762, resulting in direct association with PKCδ, which promotes cell migration. In the extracellular region, CDCP1 contains three CUB domains that are required for protein–protein interactions. CDCP1 also harbors a proteolytic cleavage (shedding) site between the first and second CUB domains and can, thus, be present in either the full-length or cleaved form depending on the cellular context. TM, transmembrane domain. (B) Schematic diagram of CRISPR/Cas9-based generation of CDCP1 -knockout MDCK cells. Blue indicates the PAM sequence and bold letters indicate the start codon. The red arrowhead indicates the cleavage site. (C) Immunoblotting analysis of CDCP1 -knockout MDCK cells. Lysates from wild-type and CDCP1 -knockout MDCK cells were subjected to immunoblotting using the indicated antibodies. (D) CDCP1 -knockout MDCK cysts were incubated in the presence of hepatocyte growth factor (50 ng/ml) for 4 and 6 d. Ki67 was visualized with an Alexa Fluor 488–conjugated antibody (green) and actin filaments were stained with Alexa Fluor 594-phalloidin (magenta). Scale bars: 10 μm. Source data are available for this figure.

    Techniques Used: Migration, CRISPR, Knock-Out, Sequencing, Western Blot, Incubation, Staining

    (A) TRE-CDCP1-EGFP (CDCP1-EGFP cells)– and mutant (YF-EGFP or CG-EGFP)–harboring MDCK cells were incubated in the presence of Dox (1 μg/ml) for the indicated time periods. Cell lysates were subjected to immunoblotting using the indicated antibodies. (B) CDCP1-EGFP cells were incubated in the presence of Dox (1 μg/ml) for the indicated time periods. Actin filaments were stained with Alexa Fluor 594-phalloidin (magenta). Scale bars indicate 50 μm. (C) CDCP1-EGFP– and mutant-harboring MDCK cells were incubated in the presence of Dox (1 μg/ml) for 48 h. Detergent-resistant membrane (DRM) and non-DRM fractions were separated on a sucrose density gradient. Aliquots of the fractions were subjected to immunoblotting analysis using the indicated antibodies. (D) DRM fractions of CDCP1-myc–overexpressing MDCK cells were subjected to immunoprecipitation with an anti-myc-tag, anti-Src, anti-Lyn, or anti-Yes antibody. Immunoprecipitates were subjected to immunoblotting using the indicated antibodies. IgG HC, IgG heavy chain. (E) A list of SFK proteins found in DRM fractions. DRM fractions of MDCK cells were subjected to SDS–PAGE and silver staining. All proteins within polyacrylamide gel were trypsinized and subjected to mass spectrometry. Proteins were identified using the SwissProt database. (F, G) DRM and non-DRM fractions of CDCP1-myc-overexpressing MDCK cells were subjected to immunoblotting analysis using the indicated antibodies. (G) Relative amount of SFK was calculated by setting the mean value for mock-transfected cells to one. Data information: In (G), the mean ratios ± SD were obtained from three independent experiments. ** P < 0.01; *** P < 0.001; NS, not significantly different; unpaired two-tailed t test. Source data are available for this figure.
    Figure Legend Snippet: (A) TRE-CDCP1-EGFP (CDCP1-EGFP cells)– and mutant (YF-EGFP or CG-EGFP)–harboring MDCK cells were incubated in the presence of Dox (1 μg/ml) for the indicated time periods. Cell lysates were subjected to immunoblotting using the indicated antibodies. (B) CDCP1-EGFP cells were incubated in the presence of Dox (1 μg/ml) for the indicated time periods. Actin filaments were stained with Alexa Fluor 594-phalloidin (magenta). Scale bars indicate 50 μm. (C) CDCP1-EGFP– and mutant-harboring MDCK cells were incubated in the presence of Dox (1 μg/ml) for 48 h. Detergent-resistant membrane (DRM) and non-DRM fractions were separated on a sucrose density gradient. Aliquots of the fractions were subjected to immunoblotting analysis using the indicated antibodies. (D) DRM fractions of CDCP1-myc–overexpressing MDCK cells were subjected to immunoprecipitation with an anti-myc-tag, anti-Src, anti-Lyn, or anti-Yes antibody. Immunoprecipitates were subjected to immunoblotting using the indicated antibodies. IgG HC, IgG heavy chain. (E) A list of SFK proteins found in DRM fractions. DRM fractions of MDCK cells were subjected to SDS–PAGE and silver staining. All proteins within polyacrylamide gel were trypsinized and subjected to mass spectrometry. Proteins were identified using the SwissProt database. (F, G) DRM and non-DRM fractions of CDCP1-myc-overexpressing MDCK cells were subjected to immunoblotting analysis using the indicated antibodies. (G) Relative amount of SFK was calculated by setting the mean value for mock-transfected cells to one. Data information: In (G), the mean ratios ± SD were obtained from three independent experiments. ** P < 0.01; *** P < 0.001; NS, not significantly different; unpaired two-tailed t test. Source data are available for this figure.

    Techniques Used: Mutagenesis, Incubation, Western Blot, Staining, Immunoprecipitation, SDS Page, Silver Staining, Mass Spectrometry, Transfection, Two Tailed Test

    (A) Schematic illustration of analysis of TRE-CDCP1-EGFP-harboring MDCK cysts (CDCP1-EGFP cysts). TRE-CDCP1-EGFP-harboring cells were cultured within a collagen matrix for 5 d for cyst formation, and then CDCP1-EGFP cysts were incubated in the presence of Dox for ∼4 d. (B) CDCP1-EGFP cysts embedded within the collagen matrix were incubated in the presence of Dox (1 μg/ml) for the indicated time periods. Arrowheads indicate protruding cells and open arrowheads indicate multi-layered structures. (C) CDCP1-EGFP cysts were incubated in the presence of Dox (1 μg/ml) for 2 d. Activated Src was visualized with a Src pY418 antibody (magenta). (D, E) CDCP1-EGFP cysts embedded within the collagen matrix were pretreated with 20 nM dasatinib or 10 μM saracatinib for 2 h, and then incubated with Dox (1 μg/ml) for 4 d. (E) Fraction of the total number of cysts counted (n > 150) with protrusions. (F, G) CDCP1-YF-EGFP and CDCP1-CG-EGFP cysts embedded within the collagen matrix were incubated in the presence of Dox (1 μg/ml) for 4 d. (G) Fraction of the total number of cysts counted (n > 150) with protrusions. (H) CDCP1-EGFP cysts embedded within the collagen matrix were pretreated with 1 mM MβCD for 2 h, and then incubated with Dox (1 μg/ml) for 4 d. Actin filaments were stained with Alexa Fluor 594-phalloidin (magenta). Scale bars: 50 μm. Data information: In (E, G), the mean ratios ± SD were obtained from three independent experiments. *** P < 0.001; **** P < 0.0001; NS, not significantly different; two-way ANOVA compared with the Dox-treated CDCP1-EGFP cysts.
    Figure Legend Snippet: (A) Schematic illustration of analysis of TRE-CDCP1-EGFP-harboring MDCK cysts (CDCP1-EGFP cysts). TRE-CDCP1-EGFP-harboring cells were cultured within a collagen matrix for 5 d for cyst formation, and then CDCP1-EGFP cysts were incubated in the presence of Dox for ∼4 d. (B) CDCP1-EGFP cysts embedded within the collagen matrix were incubated in the presence of Dox (1 μg/ml) for the indicated time periods. Arrowheads indicate protruding cells and open arrowheads indicate multi-layered structures. (C) CDCP1-EGFP cysts were incubated in the presence of Dox (1 μg/ml) for 2 d. Activated Src was visualized with a Src pY418 antibody (magenta). (D, E) CDCP1-EGFP cysts embedded within the collagen matrix were pretreated with 20 nM dasatinib or 10 μM saracatinib for 2 h, and then incubated with Dox (1 μg/ml) for 4 d. (E) Fraction of the total number of cysts counted (n > 150) with protrusions. (F, G) CDCP1-YF-EGFP and CDCP1-CG-EGFP cysts embedded within the collagen matrix were incubated in the presence of Dox (1 μg/ml) for 4 d. (G) Fraction of the total number of cysts counted (n > 150) with protrusions. (H) CDCP1-EGFP cysts embedded within the collagen matrix were pretreated with 1 mM MβCD for 2 h, and then incubated with Dox (1 μg/ml) for 4 d. Actin filaments were stained with Alexa Fluor 594-phalloidin (magenta). Scale bars: 50 μm. Data information: In (E, G), the mean ratios ± SD were obtained from three independent experiments. *** P < 0.001; **** P < 0.0001; NS, not significantly different; two-way ANOVA compared with the Dox-treated CDCP1-EGFP cysts.

    Techniques Used: Cell Culture, Incubation, Staining

    (A) Gene Ontology analysis of CDCP1-EGFP cysts. Purple bars indicate signaling related to Gene Ontology annotations. Magenta and blue bars indicate morphogenesis and cell proliferation, respectively. (B) STAT3 upstream regulatory networks of CDCP1-EGFP and CDCP1-CG-EGFP cysts. Activation Z-scores are presented in Tables S2 and S3. Red and green symbols indicate transcript levels up-regulated and down-regulated by CDCP1 overexpression, respectively. Arrows indicate activation (orange), inhibition (blue), inconsistency with the state of the downstream molecule (yellow) and unknown effect (gray). (C) Heat map representing changes in the expression of STAT3 target genes in CDCP1-EGFP (WT) and CDCP1-CG-EGFP (CG) cysts.
    Figure Legend Snippet: (A) Gene Ontology analysis of CDCP1-EGFP cysts. Purple bars indicate signaling related to Gene Ontology annotations. Magenta and blue bars indicate morphogenesis and cell proliferation, respectively. (B) STAT3 upstream regulatory networks of CDCP1-EGFP and CDCP1-CG-EGFP cysts. Activation Z-scores are presented in Tables S2 and S3. Red and green symbols indicate transcript levels up-regulated and down-regulated by CDCP1 overexpression, respectively. Arrows indicate activation (orange), inhibition (blue), inconsistency with the state of the downstream molecule (yellow) and unknown effect (gray). (C) Heat map representing changes in the expression of STAT3 target genes in CDCP1-EGFP (WT) and CDCP1-CG-EGFP (CG) cysts.

    Techniques Used: Activation Assay, Over Expression, Inhibition, Expressing

    (A) CDCP1-myc– and mutant-overexpressing MDCK cells were embedded within the collagen matrix and cultured for 9 d. Cyst lysates were subjected to immunoblotting using the indicated antibodies. (B) CDCP1-EGFP cysts were incubated with Dox (1 μg/ml) for 2 or 4 d, and then subjected to quantitative real-time PCR. Relative mRNA expression levels were calculated by setting the mean value for non-treated cysts to one. (C) CDCP1-EGFP cysts were pretreated with the indicated STAT3-specific inhibitors for 2 h and then incubated with Dox (1 μg/ml) for 4 d. (D, E) STAT3-Y705F–overexpressing CDCP1-EGFP cysts were incubated with Dox (1 μg/ml) for 4 d. (D) Actin filaments were stained with Alexa Fluor 594-phalloidin (magenta). Scale bars: 50 μm. (E) Fraction of the total number of cysts counted (n > 150) with protrusions. (F, G, H) MDCK cysts embedded within the collagen matrix were pretreated with S3i-201 (+ S3i, 50 nM; ++ S3i, 100 nM) or stattic (+ Sta, 2.5 nM; ++ Sta, 5.0 nM) for 2 h and incubated in the presence of hepatocyte growth factor (50 ng/ml) for 1 d. (F) Diameter (μm) of cysts (n = 100). (G) Ratio of Ki67-positive cell in cyst (n = 50). (H) Fraction of the total number of cysts counted (n > 100) with protrusions. Dotted blue indicates the average diameter of non-treated cysts. Data information: In (B, E, F, G, H), the mean ratios ± SD were obtained from three independent experiments. * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001; NS, not significantly different; ANOVA was calculated compared with the non-treated cyst (B) or the hepatocyte growth factor-treated cysts (F, G, H); unpaired two-tailed t test (E). Source data are available for this figure.
    Figure Legend Snippet: (A) CDCP1-myc– and mutant-overexpressing MDCK cells were embedded within the collagen matrix and cultured for 9 d. Cyst lysates were subjected to immunoblotting using the indicated antibodies. (B) CDCP1-EGFP cysts were incubated with Dox (1 μg/ml) for 2 or 4 d, and then subjected to quantitative real-time PCR. Relative mRNA expression levels were calculated by setting the mean value for non-treated cysts to one. (C) CDCP1-EGFP cysts were pretreated with the indicated STAT3-specific inhibitors for 2 h and then incubated with Dox (1 μg/ml) for 4 d. (D, E) STAT3-Y705F–overexpressing CDCP1-EGFP cysts were incubated with Dox (1 μg/ml) for 4 d. (D) Actin filaments were stained with Alexa Fluor 594-phalloidin (magenta). Scale bars: 50 μm. (E) Fraction of the total number of cysts counted (n > 150) with protrusions. (F, G, H) MDCK cysts embedded within the collagen matrix were pretreated with S3i-201 (+ S3i, 50 nM; ++ S3i, 100 nM) or stattic (+ Sta, 2.5 nM; ++ Sta, 5.0 nM) for 2 h and incubated in the presence of hepatocyte growth factor (50 ng/ml) for 1 d. (F) Diameter (μm) of cysts (n = 100). (G) Ratio of Ki67-positive cell in cyst (n = 50). (H) Fraction of the total number of cysts counted (n > 100) with protrusions. Dotted blue indicates the average diameter of non-treated cysts. Data information: In (B, E, F, G, H), the mean ratios ± SD were obtained from three independent experiments. * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001; NS, not significantly different; ANOVA was calculated compared with the non-treated cyst (B) or the hepatocyte growth factor-treated cysts (F, G, H); unpaired two-tailed t test (E). Source data are available for this figure.

    Techniques Used: Mutagenesis, Cell Culture, Western Blot, Incubation, Real-time Polymerase Chain Reaction, Expressing, Staining, Two Tailed Test

    (A) CDCP1-EGFP cysts embedded within the collagen matrix were incubated with Dox (1 μg/ml) for the indicated time periods. The basement membrane was visualized with an anti-laminin antibody (magenta). The arrowheads indicate protruding cells, and the open arrowheads indicate multi-layered structures. (B, C) CDCP1-EGFP cysts were pretreated with 20 μM marimastat for 2 h, and then incubated with Dox (1 μg/ml) for 4 d. (B) The basement membrane was visualized using an anti-laminin antibody (magenta). (C) The histogram depicting the percentage of cells with protrusions was calculated by setting the total number of cysts to 100% (n > 150). (D, E, F) MDCK cysts embedded within the collagen matrix were pretreated with marimastat (+ Mari, 10 μM; ++ Mari, 20 μM) for 2 h, and then incubated in the presence of hepatocyte growth factor (50 ng/ml) for 1 d. (D) Diameter of cysts (μm) of cysts (n = 100). (E) Ratio of Ki67-positive cell in cyst (n = 50). (F) Fraction of the total number of cysts counted (n > 100) with protrusions. Dotted blue line indicates the average diameter of non-treated cysts. (G) TRE-CDCP1-mCherry–harboring MDCK cells were embedded within a 2% DQ-collagen-containing matrix. CDCP1-mCherry cysts were pretreated with 20 μM marimastat for 2 h, and then incubated with Dox (1 μg/ml) for 4 d. DQ fluorescence (green) was visualized under a fluorescent microscope. (H) A schematic diagram of STAT3-MER protein. A modified ligand-binding domain of estrogen receptor (MER) is fused to C-terminal region of full-length STAT3 protein. SH, Src homology domain. (I, J) STAT3-MER–overexpressing cysts embedded within the collagen matrix were incubated with 1 μM 4-OHT for 4 d. (I) STAT3-MER was visualized with ERα antibody (green) and actin filaments were stained with Alexa Fluor 594-phalloidin (magenta). (J) Fraction of the total number of cysts counted (n > 100) with protrusions. Scale bars: 50 μm. Data information: In (C, D, E, F, J), the mean ratios ± SD were obtained from three independent experiments. * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001; two-way ANOVA was calculated compared with the Dox-treated cysts (C) or the hepatocyte growth factor-treated cysts (D, E, F); unpaired two-tailed t test (J).
    Figure Legend Snippet: (A) CDCP1-EGFP cysts embedded within the collagen matrix were incubated with Dox (1 μg/ml) for the indicated time periods. The basement membrane was visualized with an anti-laminin antibody (magenta). The arrowheads indicate protruding cells, and the open arrowheads indicate multi-layered structures. (B, C) CDCP1-EGFP cysts were pretreated with 20 μM marimastat for 2 h, and then incubated with Dox (1 μg/ml) for 4 d. (B) The basement membrane was visualized using an anti-laminin antibody (magenta). (C) The histogram depicting the percentage of cells with protrusions was calculated by setting the total number of cysts to 100% (n > 150). (D, E, F) MDCK cysts embedded within the collagen matrix were pretreated with marimastat (+ Mari, 10 μM; ++ Mari, 20 μM) for 2 h, and then incubated in the presence of hepatocyte growth factor (50 ng/ml) for 1 d. (D) Diameter of cysts (μm) of cysts (n = 100). (E) Ratio of Ki67-positive cell in cyst (n = 50). (F) Fraction of the total number of cysts counted (n > 100) with protrusions. Dotted blue line indicates the average diameter of non-treated cysts. (G) TRE-CDCP1-mCherry–harboring MDCK cells were embedded within a 2% DQ-collagen-containing matrix. CDCP1-mCherry cysts were pretreated with 20 μM marimastat for 2 h, and then incubated with Dox (1 μg/ml) for 4 d. DQ fluorescence (green) was visualized under a fluorescent microscope. (H) A schematic diagram of STAT3-MER protein. A modified ligand-binding domain of estrogen receptor (MER) is fused to C-terminal region of full-length STAT3 protein. SH, Src homology domain. (I, J) STAT3-MER–overexpressing cysts embedded within the collagen matrix were incubated with 1 μM 4-OHT for 4 d. (I) STAT3-MER was visualized with ERα antibody (green) and actin filaments were stained with Alexa Fluor 594-phalloidin (magenta). (J) Fraction of the total number of cysts counted (n > 100) with protrusions. Scale bars: 50 μm. Data information: In (C, D, E, F, J), the mean ratios ± SD were obtained from three independent experiments. * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001; two-way ANOVA was calculated compared with the Dox-treated cysts (C) or the hepatocyte growth factor-treated cysts (D, E, F); unpaired two-tailed t test (J).

    Techniques Used: Incubation, Fluorescence, Microscopy, Modification, Ligand Binding Assay, Staining, Two Tailed Test

    (A) Wild-type and CDCP1 -knockout TRE-Met harboring MDCK cells embedded within the collagen matrix were incubated with hepatocyte growth factor (50 ng/ml) or Dox (1 μg/ml) for 4 d. Met was visualized with a Met antibody (green) and actin filaments were stained with Alexa Fluor 594-phalloidin (magenta). Scale bars indicate 50 μm. (B) Renal cancer cell lines, A498 and ACHN, were incubated in the presence or absence of hepatocyte growth factor (50 ng/ml) for 30 min. Cell lysates were subjected to immunoprecipitation with anti-CDCP1 or anti-Met antibody. Immunoprecipitates were subjected to immunoblotting using the indicated antibodies. IgG HC, IgG heavy chain. Source data are available for this figure.
    Figure Legend Snippet: (A) Wild-type and CDCP1 -knockout TRE-Met harboring MDCK cells embedded within the collagen matrix were incubated with hepatocyte growth factor (50 ng/ml) or Dox (1 μg/ml) for 4 d. Met was visualized with a Met antibody (green) and actin filaments were stained with Alexa Fluor 594-phalloidin (magenta). Scale bars indicate 50 μm. (B) Renal cancer cell lines, A498 and ACHN, were incubated in the presence or absence of hepatocyte growth factor (50 ng/ml) for 30 min. Cell lysates were subjected to immunoprecipitation with anti-CDCP1 or anti-Met antibody. Immunoprecipitates were subjected to immunoblotting using the indicated antibodies. IgG HC, IgG heavy chain. Source data are available for this figure.

    Techniques Used: Knock-Out, Incubation, Staining, Immunoprecipitation, Western Blot

    (A) CDCP1-EGFP cysts were pretreated with the indicated Met-specific inhibitors for 2 h and then incubated with Dox (1 μg/ml) for 4 d. (B, C) Schematic representation of CDCP1 and the deletion mutants. (B) SP, signal peptide; CUB, CUB domain; TM, transmembrane domain; CD, cytosolic domain. Lysates from HEK293 cells overexpressing both CDCP1-myc and Met were subjected to immunoprecipitation with an anti-myc-tag antibody. Immunoprecipitates were subjected to immunoblotting using the indicated antibodies. IgG HC, IgG heavy chain. (D) CDCP1-myc- and CDCP1-PR-myc-overexpressing MDCK cells were embedded within the collagen matrix and cultured for 9 d. Cyst lysates were subjected to immunoblotting analysis using the indicated antibodies. (E, F) CDCP1-PR-EGFP cysts embedded within the collagen matrix were incubated with Dox (1 μg/ml) for 4 d. (E) Actin filaments were stained with Alexa Fluor 594-phalloidin (magenta). Scale bars: 50 μm. (F) Fraction of the total number of cysts counted (n > 150) with protrusions. Data information: In (F), the mean ratios ± SD were obtained from three independent experiments. ** P < 0.01; **** P < 0.0001; NS, not significantly different; two-way ANOVA was calculated compared with the Dox-treated cysts. Source data are available for this figure.
    Figure Legend Snippet: (A) CDCP1-EGFP cysts were pretreated with the indicated Met-specific inhibitors for 2 h and then incubated with Dox (1 μg/ml) for 4 d. (B, C) Schematic representation of CDCP1 and the deletion mutants. (B) SP, signal peptide; CUB, CUB domain; TM, transmembrane domain; CD, cytosolic domain. Lysates from HEK293 cells overexpressing both CDCP1-myc and Met were subjected to immunoprecipitation with an anti-myc-tag antibody. Immunoprecipitates were subjected to immunoblotting using the indicated antibodies. IgG HC, IgG heavy chain. (D) CDCP1-myc- and CDCP1-PR-myc-overexpressing MDCK cells were embedded within the collagen matrix and cultured for 9 d. Cyst lysates were subjected to immunoblotting analysis using the indicated antibodies. (E, F) CDCP1-PR-EGFP cysts embedded within the collagen matrix were incubated with Dox (1 μg/ml) for 4 d. (E) Actin filaments were stained with Alexa Fluor 594-phalloidin (magenta). Scale bars: 50 μm. (F) Fraction of the total number of cysts counted (n > 150) with protrusions. Data information: In (F), the mean ratios ± SD were obtained from three independent experiments. ** P < 0.01; **** P < 0.0001; NS, not significantly different; two-way ANOVA was calculated compared with the Dox-treated cysts. Source data are available for this figure.

    Techniques Used: Incubation, Immunoprecipitation, Western Blot, Cell Culture, Staining

    (A) CDCP1-EGFP, CDCP1-YF-EGFP, and CDCP1-PR-EGFP cells were incubated with Dox (1 μg/ml) for 2, 4 d. Cell lysates were subjected to immunoblotting analysis using the indicated analysis using the indicated antibodies. (B, C) CDCP1-EGFP cells were incubated with Dox (1 μg/ml) for 2, 4 d. NK4 (1 μg/ml) were added 2 h before the Dox treatment. Saracatinib (10 μM, Sara) or Met/Ron kinase inhibitor (100 nM, Met inh.) were added 3 d after the Dox treatment. Met were stained with Alexa Fluor 594-phalloidin (magenta). Scale bars: 20 μm. (C) Ratio of plasma membrane-localized Met was calculated (n = 50). (D) CDCP1-EGFP cells were incubated with Dox (1 μg/ml) for 4 d. NK4 (1 μg/ml) were added 2 h before the Dox treatment. Saracatinib (+ Sara, 10 μM; ++ Sara 20 μM) were added 3 d after the Dox treatment. Cell lysates were subjected to immunoblotting analysis using the indicated analysis using the indicated antibodies. (E, F) CDCP1-EGFP cells were incubated with Dox (1 μg/ml) for 4 d. Saracatinib (10 μM, Sara) were added 3 d after the Dox treatment. Met/Ron kinase inhibitor (100 nM, Met inh.) were added 30 min before cell extraction. Cell lysates were subjected to immunoprecipitation with an anti-Met antibody, and immunoprecipitates were subjected to immunoblotting using the indicated antibodies. IgG HC, IgG heavy chain. (F) Relative ratio of associated STAT3 was calculated by setting the mean value for non-treated cells to one. (G) Schematic diagram of the CDCP1-Src-Met complex-mediated STAT3 activation. Data information: In (C, F), the mean ratios ± SD were obtained from three independent experiments. * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001; NS, not significantly different; two-way ANOVA was calculated compared with the Dox-treated cells (C); unpaired two-tailed t test (F). Source data are available for this figure.
    Figure Legend Snippet: (A) CDCP1-EGFP, CDCP1-YF-EGFP, and CDCP1-PR-EGFP cells were incubated with Dox (1 μg/ml) for 2, 4 d. Cell lysates were subjected to immunoblotting analysis using the indicated analysis using the indicated antibodies. (B, C) CDCP1-EGFP cells were incubated with Dox (1 μg/ml) for 2, 4 d. NK4 (1 μg/ml) were added 2 h before the Dox treatment. Saracatinib (10 μM, Sara) or Met/Ron kinase inhibitor (100 nM, Met inh.) were added 3 d after the Dox treatment. Met were stained with Alexa Fluor 594-phalloidin (magenta). Scale bars: 20 μm. (C) Ratio of plasma membrane-localized Met was calculated (n = 50). (D) CDCP1-EGFP cells were incubated with Dox (1 μg/ml) for 4 d. NK4 (1 μg/ml) were added 2 h before the Dox treatment. Saracatinib (+ Sara, 10 μM; ++ Sara 20 μM) were added 3 d after the Dox treatment. Cell lysates were subjected to immunoblotting analysis using the indicated analysis using the indicated antibodies. (E, F) CDCP1-EGFP cells were incubated with Dox (1 μg/ml) for 4 d. Saracatinib (10 μM, Sara) were added 3 d after the Dox treatment. Met/Ron kinase inhibitor (100 nM, Met inh.) were added 30 min before cell extraction. Cell lysates were subjected to immunoprecipitation with an anti-Met antibody, and immunoprecipitates were subjected to immunoblotting using the indicated antibodies. IgG HC, IgG heavy chain. (F) Relative ratio of associated STAT3 was calculated by setting the mean value for non-treated cells to one. (G) Schematic diagram of the CDCP1-Src-Met complex-mediated STAT3 activation. Data information: In (C, F), the mean ratios ± SD were obtained from three independent experiments. * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001; NS, not significantly different; two-way ANOVA was calculated compared with the Dox-treated cells (C); unpaired two-tailed t test (F). Source data are available for this figure.

    Techniques Used: Incubation, Western Blot, Staining, Immunoprecipitation, Activation Assay, Two Tailed Test

    (A, B, C) Related to . Relative ratio of expression (A) and activation level of Met (B, C) was calculated by setting the mean value for non-treated cells to one. (D) CDCP1-EGFP and CDCP1-YF-EGFP cells were incubated with Dox (1 μg/ml) for 4 d. Detergent-resistant membrane and non-detergent-resistant membrane fractions were separated in a sucrose density gradient. Aliquots of the fractions were subjected to immunoblotting analysis using the indicated antibodies. (E, F, G) Related to . Relative ratio of expression (E) and activation level of Met (F, G) was calculated by setting the mean value for Dox-treated cells to one. (H) A schematic model of the role of CDCP1-Src in hepatocyte growth factor-induced morphogenesis. CDCP1-Src activates the Met-STAT3 signaling on lipid rafts, leading to outgrowth through induction of ECM rearrangement and cell growth/proliferation. The mammalian target of rapamycin pathway contributes to cell growth. (I, J, K) CDCP1-EGFP cells were incubated in medium supplemented with the presence of indicated FBS concentration with Dox (1 μg/ml) for 4 d. (I) Cell lysates were subjected to immunoblotting using the indicated antibodies. (J, K) Relative ratio of Met phosphorylation level was calculated by setting the mean value for non-treated cells to one. Data information: In (A, B, C, E, F, G, J, K), the mean ratios ± SD were obtained from at least three independent experiments. NS, not significantly different; two-way ANOVA was calculated compared with the non–Dox-treated cells (A, B, C) or the Dox-treated cells (E, F, G); unpaired two-tailed t test (J, K). Source data are available for this figure.
    Figure Legend Snippet: (A, B, C) Related to . Relative ratio of expression (A) and activation level of Met (B, C) was calculated by setting the mean value for non-treated cells to one. (D) CDCP1-EGFP and CDCP1-YF-EGFP cells were incubated with Dox (1 μg/ml) for 4 d. Detergent-resistant membrane and non-detergent-resistant membrane fractions were separated in a sucrose density gradient. Aliquots of the fractions were subjected to immunoblotting analysis using the indicated antibodies. (E, F, G) Related to . Relative ratio of expression (E) and activation level of Met (F, G) was calculated by setting the mean value for Dox-treated cells to one. (H) A schematic model of the role of CDCP1-Src in hepatocyte growth factor-induced morphogenesis. CDCP1-Src activates the Met-STAT3 signaling on lipid rafts, leading to outgrowth through induction of ECM rearrangement and cell growth/proliferation. The mammalian target of rapamycin pathway contributes to cell growth. (I, J, K) CDCP1-EGFP cells were incubated in medium supplemented with the presence of indicated FBS concentration with Dox (1 μg/ml) for 4 d. (I) Cell lysates were subjected to immunoblotting using the indicated antibodies. (J, K) Relative ratio of Met phosphorylation level was calculated by setting the mean value for non-treated cells to one. Data information: In (A, B, C, E, F, G, J, K), the mean ratios ± SD were obtained from at least three independent experiments. NS, not significantly different; two-way ANOVA was calculated compared with the non–Dox-treated cells (A, B, C) or the Dox-treated cells (E, F, G); unpaired two-tailed t test (J, K). Source data are available for this figure.

    Techniques Used: Expressing, Activation Assay, Incubation, Western Blot, Concentration Assay, Two Tailed Test

    (A) Schematic diagram of CRISPR/Cas9-based generation of Cdcp1 -knockout mouse. Blue indicates the PAM sequence and bold letters indicate the start codon. The red arrowhead indicates the cleavage site. (B) Representative picture of wild-type (+/+), Cdcp1 heterozygous (+/−) and homozygous knockout (−/−) littermate mice at 8 wk of age. (C, D) Validation of gene knockout was performed by genotyping (C) and Western blot (D). PCR verification of deletion at first exon of Cdcp1 gene using Fw and Rev primers depicted in panel (A). Lysates from mouse kidney were subjected to immunoblotting using the indicated antibodies. (E, F) Whole body (E) and kidney (F) weight of wild-type (+/+) and Cdcp1 homozygous knockout (−/−) mice at 8 wk of age. Data information: In (E, F), the mean ratios ± SD were obtained from 10 mice per group. NS, not significantly different; unpaired two-tailed t test. Source data are available for this figure.
    Figure Legend Snippet: (A) Schematic diagram of CRISPR/Cas9-based generation of Cdcp1 -knockout mouse. Blue indicates the PAM sequence and bold letters indicate the start codon. The red arrowhead indicates the cleavage site. (B) Representative picture of wild-type (+/+), Cdcp1 heterozygous (+/−) and homozygous knockout (−/−) littermate mice at 8 wk of age. (C, D) Validation of gene knockout was performed by genotyping (C) and Western blot (D). PCR verification of deletion at first exon of Cdcp1 gene using Fw and Rev primers depicted in panel (A). Lysates from mouse kidney were subjected to immunoblotting using the indicated antibodies. (E, F) Whole body (E) and kidney (F) weight of wild-type (+/+) and Cdcp1 homozygous knockout (−/−) mice at 8 wk of age. Data information: In (E, F), the mean ratios ± SD were obtained from 10 mice per group. NS, not significantly different; unpaired two-tailed t test. Source data are available for this figure.

    Techniques Used: CRISPR, Knock-Out, Sequencing, Gene Knockout, Western Blot, Two Tailed Test

    (A, B) Cdcp1 wild-type (+/+), heterozygous (+/−), and homozygous knockout (−/−) mice at 8 wk of age were subjected to UNX or sham operation. Regenerative growth of remaining kidney was analyzed 8 wk after operation and increases in remaining kidney/body weight ratios were assessed. (C) The remaining kidney was removed from UNX- or sham-operated mice, and was subjected to hematoxylin-eosin (HE) staining; arrowheads indicate glomeruli. (D, E, F) The remaining kidney after UNX was subjected to microscopic immunofluorescence analysis with specific antibodies against collagen IV (magenta), and proximal tubules were visualized with FITC-LTL (green). Scale bars: 50 μm. Thickness (E) and area (F) of proximal tubules were determined by the distance (μm, n = 100) and area (μm 2 , n = 50) between FITC-LTL-stained lumen and collagen IV-enriched basement membrane as depicted in panel (D). (G) Area (μm 2 ) of glomerulus in the remaining kidney was evaluated by using HE-staining images in panel (C) (n = 50). Data information: In (B, E, F, G), the mean ratios ± SD were obtained from 10 (B) or five mice (E, F, G) per group. * P < 0.05; ** P < 0.01; **** P < 0.0001; NS, not significantly different; two-way ANOVA.
    Figure Legend Snippet: (A, B) Cdcp1 wild-type (+/+), heterozygous (+/−), and homozygous knockout (−/−) mice at 8 wk of age were subjected to UNX or sham operation. Regenerative growth of remaining kidney was analyzed 8 wk after operation and increases in remaining kidney/body weight ratios were assessed. (C) The remaining kidney was removed from UNX- or sham-operated mice, and was subjected to hematoxylin-eosin (HE) staining; arrowheads indicate glomeruli. (D, E, F) The remaining kidney after UNX was subjected to microscopic immunofluorescence analysis with specific antibodies against collagen IV (magenta), and proximal tubules were visualized with FITC-LTL (green). Scale bars: 50 μm. Thickness (E) and area (F) of proximal tubules were determined by the distance (μm, n = 100) and area (μm 2 , n = 50) between FITC-LTL-stained lumen and collagen IV-enriched basement membrane as depicted in panel (D). (G) Area (μm 2 ) of glomerulus in the remaining kidney was evaluated by using HE-staining images in panel (C) (n = 50). Data information: In (B, E, F, G), the mean ratios ± SD were obtained from 10 (B) or five mice (E, F, G) per group. * P < 0.05; ** P < 0.01; **** P < 0.0001; NS, not significantly different; two-way ANOVA.

    Techniques Used: Knock-Out, Staining, Immunofluorescence

    (A, B, C) Related to . Cdcp1 wild-type (+/+) and homozygous knockout (−/−) mice at 8 wk of age were subjected to UNX or sham operation. After 8 wk, weight of body (A) and the remaining kidney (B) were measured, and UNX-induced percent increase in remaining kidney/body weight ratios were assessed (C). (D) Related to . Cdcp1 wild-type (+/+), heterozygous (+/−), and homozygous knockout (−/−) mice at 8 wk of age were subjected to UNX or sham operation. The remaining kidney was removed from UNX- or sham-operated mice, and was subjected to hematoxylin-eosin (HE) staining. Scale bars indicate 1,000 μm. Data information: In (A, B, C), the mean ratios ± SD were obtained from at least 13 mice per group. * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001; NS, not significantly different; two-way ANOVA (A, B); unpaired two-tailed t test (C).
    Figure Legend Snippet: (A, B, C) Related to . Cdcp1 wild-type (+/+) and homozygous knockout (−/−) mice at 8 wk of age were subjected to UNX or sham operation. After 8 wk, weight of body (A) and the remaining kidney (B) were measured, and UNX-induced percent increase in remaining kidney/body weight ratios were assessed (C). (D) Related to . Cdcp1 wild-type (+/+), heterozygous (+/−), and homozygous knockout (−/−) mice at 8 wk of age were subjected to UNX or sham operation. The remaining kidney was removed from UNX- or sham-operated mice, and was subjected to hematoxylin-eosin (HE) staining. Scale bars indicate 1,000 μm. Data information: In (A, B, C), the mean ratios ± SD were obtained from at least 13 mice per group. * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001; NS, not significantly different; two-way ANOVA (A, B); unpaired two-tailed t test (C).

    Techniques Used: Knock-Out, Staining, Two Tailed Test

    (A, B) Cdcp1 wild-type (+/+) and homozygous knockout (−/−) mice at 8 wk of age were subjected to UNX or sham operation. Compensatory growth of remaining kidney was analyzed at the indicated time points after operation and increases in remaining kidney/body weight ratios were assessed. (C, D, E, F) The remaining kidney after UNX was subjected to microscopic immunofluorescence analysis with specific antibodies against Met pY1234/1235 (C), STAT3 pY705 (D), CDCP1 pY734 (E), and Ki67 (F) and Alexa Fluor 594–conjugated secondary antibody (magenta). Proximal tubules were visualized by staining with FITC-LTL (green). Representative images were shown. Scale bars: 50 μm. (G) Ki67-positive proximal tubules (%) were estimated by calculating the ratio of Ki67-stained tubules to the total number of tubules (n > 100). (H) Schematic model of hepatocyte growth factor-induced adaptive renal regeneration. CDCP1-Src regulates the Met-STAT3 signaling leading to compensatory renal growth through induction of ECM rearrangement and cell growth/proliferation. Data information: In (A, G), the mean ratios ± SD were obtained from at least six (A) or three mice (G) per group. * P < 0.05; ** P < 0.01; **** P < 0.0001; NS, not significantly different; two-way ANOVA was calculated compared with sham-operated control or between wild-type and knockout mice.
    Figure Legend Snippet: (A, B) Cdcp1 wild-type (+/+) and homozygous knockout (−/−) mice at 8 wk of age were subjected to UNX or sham operation. Compensatory growth of remaining kidney was analyzed at the indicated time points after operation and increases in remaining kidney/body weight ratios were assessed. (C, D, E, F) The remaining kidney after UNX was subjected to microscopic immunofluorescence analysis with specific antibodies against Met pY1234/1235 (C), STAT3 pY705 (D), CDCP1 pY734 (E), and Ki67 (F) and Alexa Fluor 594–conjugated secondary antibody (magenta). Proximal tubules were visualized by staining with FITC-LTL (green). Representative images were shown. Scale bars: 50 μm. (G) Ki67-positive proximal tubules (%) were estimated by calculating the ratio of Ki67-stained tubules to the total number of tubules (n > 100). (H) Schematic model of hepatocyte growth factor-induced adaptive renal regeneration. CDCP1-Src regulates the Met-STAT3 signaling leading to compensatory renal growth through induction of ECM rearrangement and cell growth/proliferation. Data information: In (A, G), the mean ratios ± SD were obtained from at least six (A) or three mice (G) per group. * P < 0.05; ** P < 0.01; **** P < 0.0001; NS, not significantly different; two-way ANOVA was calculated compared with sham-operated control or between wild-type and knockout mice.

    Techniques Used: Knock-Out, Immunofluorescence, Staining

    (A, B, C) The remaining kidney of wild-type ( Cdcp1 +/+) mice after UNX was subjected to immunofluorescence microscopic analysis using specific antibodies against Met pY1234/1235 (A), STAT3 pY705 (B), or Src pY418 (C) and Alexa Fluor 488-conjugated secondary antibody (green). Endosome marker EEA1 was visualized using Alexa Fluor 594–conjugated secondary antibody (magenta). (D, E, F, G) The remaining kidney of Cdcp1 wild-type (+/+) and Cdcp1 homozygous knockout (−/−) mice after UNX was subjected to immunofluorescence microscopic analysis using specific antibodies against collagen IV (D), MMP2 (F), or MMP9 (G) and Alexa Fluor 594–conjugated secondary antibody (magenta). Proximal tubules were visualized by staining with FITC-LTL (green). Representative images were shown. Scale bars indicate 50 μm. (E) Relative intensity of collagen IV surrounding FITC-LTL–positive proximal tubule (n = 50) were calculated by setting the mean value for sham-operated wild-type mice to one. See also . (H) Schematic diagram of adaptive renal regeneration of wild-type (+/+) and Cdcp1 homozygous knockout (−/−) mice. Data information: In (E), the mean ratios ± SD were obtained from five mice per group. * P < 0.05; ** P < 0.01; NS, not significantly different; two-way ANOVA.
    Figure Legend Snippet: (A, B, C) The remaining kidney of wild-type ( Cdcp1 +/+) mice after UNX was subjected to immunofluorescence microscopic analysis using specific antibodies against Met pY1234/1235 (A), STAT3 pY705 (B), or Src pY418 (C) and Alexa Fluor 488-conjugated secondary antibody (green). Endosome marker EEA1 was visualized using Alexa Fluor 594–conjugated secondary antibody (magenta). (D, E, F, G) The remaining kidney of Cdcp1 wild-type (+/+) and Cdcp1 homozygous knockout (−/−) mice after UNX was subjected to immunofluorescence microscopic analysis using specific antibodies against collagen IV (D), MMP2 (F), or MMP9 (G) and Alexa Fluor 594–conjugated secondary antibody (magenta). Proximal tubules were visualized by staining with FITC-LTL (green). Representative images were shown. Scale bars indicate 50 μm. (E) Relative intensity of collagen IV surrounding FITC-LTL–positive proximal tubule (n = 50) were calculated by setting the mean value for sham-operated wild-type mice to one. See also . (H) Schematic diagram of adaptive renal regeneration of wild-type (+/+) and Cdcp1 homozygous knockout (−/−) mice. Data information: In (E), the mean ratios ± SD were obtained from five mice per group. * P < 0.05; ** P < 0.01; NS, not significantly different; two-way ANOVA.

    Techniques Used: Immunofluorescence, Marker, Knock-Out, Staining

    (A) Down-regulation of CDCP1 induces formation of a luminal structure. CDCP1-EGFP cysts embedded within the collagen matrix were incubated in the presence of Dox (1 μg/ml) for 4 d. Cysts were then incubated for an additional 5 d in the absence of Dox. (B) Contribution of mammalian target of rapamycin pathway and STAT3-MMP axis. CDCP1-EGFP cysts were pretreated with Torin1 (50 nM) or rapamycin (Rapa, 200 nM) and marimastat (Mari, 20 μM) for 2 h and then incubated with Dox (1 μg/ml) for 4 d. (C, D) Contribution of PKCδ signaling. (C) CDCP1-Y762F-EGFP cysts embedded within the collagen matrix were incubated in the presence of Dox (1 μg/ml) for 4 d. (D) CDCP1-EGFP cysts were pretreated with Gö6983 for 2 h and then incubated with Dox (1 μg/ml) for 4 d. (E, F) Analysis of hepatocyte growth factor dependency. CDCP1-EGFP cysts were pretreated with NK4 (1 μg/ml) for 2 h and then incubated with Dox (1 μg/ml) for 4 d. (E) Actin filaments were stained with Alexa Fluor 594-phalloidin (magenta). (F) Fraction of the total number of cysts counted (n > 100) with protrusions. (G) Up-regulation of CDCP1 induces an expression of cytokeratin 14 (CK14). TRE-CDCP1-EGFP-harboring cysts were incubated with Dox (1 μg/ml) for 4 d. Cytokeratin 14 was visualized with a specific antibody (magenta). Scale bars indicate 50 μm. Data information: In (F), the mean ratios ± SD were obtained from three independent experiments. NS, not significantly different; unpaired two-tailed t test.
    Figure Legend Snippet: (A) Down-regulation of CDCP1 induces formation of a luminal structure. CDCP1-EGFP cysts embedded within the collagen matrix were incubated in the presence of Dox (1 μg/ml) for 4 d. Cysts were then incubated for an additional 5 d in the absence of Dox. (B) Contribution of mammalian target of rapamycin pathway and STAT3-MMP axis. CDCP1-EGFP cysts were pretreated with Torin1 (50 nM) or rapamycin (Rapa, 200 nM) and marimastat (Mari, 20 μM) for 2 h and then incubated with Dox (1 μg/ml) for 4 d. (C, D) Contribution of PKCδ signaling. (C) CDCP1-Y762F-EGFP cysts embedded within the collagen matrix were incubated in the presence of Dox (1 μg/ml) for 4 d. (D) CDCP1-EGFP cysts were pretreated with Gö6983 for 2 h and then incubated with Dox (1 μg/ml) for 4 d. (E, F) Analysis of hepatocyte growth factor dependency. CDCP1-EGFP cysts were pretreated with NK4 (1 μg/ml) for 2 h and then incubated with Dox (1 μg/ml) for 4 d. (E) Actin filaments were stained with Alexa Fluor 594-phalloidin (magenta). (F) Fraction of the total number of cysts counted (n > 100) with protrusions. (G) Up-regulation of CDCP1 induces an expression of cytokeratin 14 (CK14). TRE-CDCP1-EGFP-harboring cysts were incubated with Dox (1 μg/ml) for 4 d. Cytokeratin 14 was visualized with a specific antibody (magenta). Scale bars indicate 50 μm. Data information: In (F), the mean ratios ± SD were obtained from three independent experiments. NS, not significantly different; unpaired two-tailed t test.

    Techniques Used: Incubation, Staining, Expressing, Two Tailed Test

    anti cdcp1 py734 antibody  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc anti cdcp1 py734 antibody
    Anti Cdcp1 Py734 Antibody, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    cdcp1 py734  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc cdcp1 py734
    Antibodies and Dilutions Used for IHC and Western Blot Analysis
    Cdcp1 Py734, 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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    1) Product Images from "Src Kinase Is Biphosphorylated at Y416/Y527 and Activates the CUB-Domain Containing Protein 1/Protein Kinase C δ Pathway in a Subset of Triple-Negative Breast Cancers"

    Article Title: Src Kinase Is Biphosphorylated at Y416/Y527 and Activates the CUB-Domain Containing Protein 1/Protein Kinase C δ Pathway in a Subset of Triple-Negative Breast Cancers

    Journal: The American Journal of Pathology

    doi: 10.1016/j.ajpath.2019.10.017

    Antibodies and Dilutions Used for IHC and Western Blot Analysis
    Figure Legend Snippet: Antibodies and Dilutions Used for IHC and Western Blot Analysis

    Techniques Used: Western Blot

    SFK substrates CUB-domain containing protein 1 (CDCP1) and protein kinase C δ (PKCδ) are phosphorylated in SFK_pY416 positive triple-negative breast cancer (TNBC). A: Representative patient sample with diffuse immunohistochemical staining of tumor cells for SFK_pY416, CDCP1_pY743, and PKCä_pY311. Staining for each marker was detected primarily on the cell membrane. Negative cells are predominantly stromal lymphocytes. B: Representative patient sample with a focal/low (<20%) pattern of staining for SFK_pY416, CDCP1_pY743, and PKCä_pY311 in tumor cells. C: Representative patient sample with negative staining. Insets: Areas of interest at higher magnification to show detail. D: Summary of staining results in panel of 56 TNBC samples. Main images were acquired using a 10× objective, and insets were acquired using a 40× objective. Scale bars = 100 μm (A–C). FOXA, forkhead box protein.
    Figure Legend Snippet: SFK substrates CUB-domain containing protein 1 (CDCP1) and protein kinase C δ (PKCδ) are phosphorylated in SFK_pY416 positive triple-negative breast cancer (TNBC). A: Representative patient sample with diffuse immunohistochemical staining of tumor cells for SFK_pY416, CDCP1_pY743, and PKCä_pY311. Staining for each marker was detected primarily on the cell membrane. Negative cells are predominantly stromal lymphocytes. B: Representative patient sample with a focal/low (<20%) pattern of staining for SFK_pY416, CDCP1_pY743, and PKCä_pY311 in tumor cells. C: Representative patient sample with negative staining. Insets: Areas of interest at higher magnification to show detail. D: Summary of staining results in panel of 56 TNBC samples. Main images were acquired using a 10× objective, and insets were acquired using a 40× objective. Scale bars = 100 μm (A–C). FOXA, forkhead box protein.

    Techniques Used: Immunohistochemical staining, Staining, Marker, Negative Staining

    Clinical and Pathologic Features of TNBC Patient Samples in Relation to High (> 20%) SFK_pY416 Expression
    Figure Legend Snippet: Clinical and Pathologic Features of TNBC Patient Samples in Relation to High (> 20%) SFK_pY416 Expression

    Techniques Used:

    SFK_pY416 and SFK_pY527 are co-expressed in triple-negative breast cancer (TNBC). A: Western blot analysis confirms the co-expression of phosphorylated SFK/CUB-domain containing protein 1 (CDCP1) proteins, as determined by immunohistochemistry (IHC), and shows the co-expression of SFK_pY416 and SFK_pY527. Paraffin sections of TNBC were extracted and analyzed by standard Western blot analysis technique. forkhead box protein 1 (FOXA1), vimentin, and SFK_pY416 expression levels in tumor cells, as determined by IHC, are summarized on top. Probing with SFK_pY416 antibody detects a 60-kDa band in all patient samples that were SFK_pY416+ by IHC (lanes 4 and 8 to 13) and does not detect the band in patient samples that were SFK_pY416− by IHC. Cleaved CDCP1_pY734, CDCP1_pY743, and SFK_pY527 are co-expressed with SFK_pY416. Whole cell lysate from the TNBC cell line MDA-MB-231 was used as a positive control (lane 1). TNBC-2 (lane 4) has granular cytoplasmic staining for SFK_pY416. All other SFK_pY416+ tumors had membrane staining. Equal amounts of protein were loaded to each lane, as quantitated by tryptophan fluorescence spectrometry. β-Actin was used as a loading control. B: Analysis of TNBC proteome in silico showing top eight proteins (protein names displayed next to data point) whose expression is correlated with SFK_pY416, with SFK_pY527 exhibiting the strongest correlation. TNBC tumor samples were isolated from The Cancer Proteome Atlas (TCPA) breast cancer (BRCA) data set by negativity for estrogen receptor, progesterone receptor, and human epidermal growth factor 2 receptor and analyzed for correlation of protein markers. Correlations were derived by Pearson product moment correlation test. cCDCP1, cleaved CDCP1; EGFR, epidermal growth factor receptor; FASN, fatty acid synthase; flCDCP1, full-length CDCP1; MEK1, dual specificity mitogen-activated protein kinase kinase 1; NFKBp65, nuclear factor NF-kappa-B p65 subunit; PEA15, astrocytic phosphoprotein PEA-15; RAB25, Ras-related protein rab-25; SFK, Src family kinase; SHP2, Src homology region 2–containing protein tyrosine phosphatase; STAT3, signal transducer and activator of transcription 3.
    Figure Legend Snippet: SFK_pY416 and SFK_pY527 are co-expressed in triple-negative breast cancer (TNBC). A: Western blot analysis confirms the co-expression of phosphorylated SFK/CUB-domain containing protein 1 (CDCP1) proteins, as determined by immunohistochemistry (IHC), and shows the co-expression of SFK_pY416 and SFK_pY527. Paraffin sections of TNBC were extracted and analyzed by standard Western blot analysis technique. forkhead box protein 1 (FOXA1), vimentin, and SFK_pY416 expression levels in tumor cells, as determined by IHC, are summarized on top. Probing with SFK_pY416 antibody detects a 60-kDa band in all patient samples that were SFK_pY416+ by IHC (lanes 4 and 8 to 13) and does not detect the band in patient samples that were SFK_pY416− by IHC. Cleaved CDCP1_pY734, CDCP1_pY743, and SFK_pY527 are co-expressed with SFK_pY416. Whole cell lysate from the TNBC cell line MDA-MB-231 was used as a positive control (lane 1). TNBC-2 (lane 4) has granular cytoplasmic staining for SFK_pY416. All other SFK_pY416+ tumors had membrane staining. Equal amounts of protein were loaded to each lane, as quantitated by tryptophan fluorescence spectrometry. β-Actin was used as a loading control. B: Analysis of TNBC proteome in silico showing top eight proteins (protein names displayed next to data point) whose expression is correlated with SFK_pY416, with SFK_pY527 exhibiting the strongest correlation. TNBC tumor samples were isolated from The Cancer Proteome Atlas (TCPA) breast cancer (BRCA) data set by negativity for estrogen receptor, progesterone receptor, and human epidermal growth factor 2 receptor and analyzed for correlation of protein markers. Correlations were derived by Pearson product moment correlation test. cCDCP1, cleaved CDCP1; EGFR, epidermal growth factor receptor; FASN, fatty acid synthase; flCDCP1, full-length CDCP1; MEK1, dual specificity mitogen-activated protein kinase kinase 1; NFKBp65, nuclear factor NF-kappa-B p65 subunit; PEA15, astrocytic phosphoprotein PEA-15; RAB25, Ras-related protein rab-25; SFK, Src family kinase; SHP2, Src homology region 2–containing protein tyrosine phosphatase; STAT3, signal transducer and activator of transcription 3.

    Techniques Used: Western Blot, Expressing, Immunohistochemistry, Positive Control, Staining, Fluorescence, In Silico, Isolation, Derivative Assay

    Western blot analysis of SFK_pY416− triple-negative breast cancer (TNBC) reveals that forkhead box protein 1 (FOXA1)+ samples tend to be SFK_pY527+ and FOXA1− samples tend to be SFK_pY527−. Paraffin sections of TNBC were extracted and analyzed by standard Western blot analysis technique. FOXA1, vimentin, and SFK_pY416 expression levels in tumor cells, as determined by immunohistochemistry (IHC), are summarized on top. A: All FOXA1+/SFK_pY416− samples tested were SFK_pY527+. The presence of SFK_pY416 in lanes 2, 4, and 6 is due to the presence of SFK_pY416+ lymphocytes in these tumor samples (shown in Supplemental Figure S1). B: Five of six FOXA1−/SFK_pY416− samples tested were SFK_pY527− (lanes 2, 3, and 5 to 7). Three FOXA1−/SFK_pY416+ tumor samples were run as controls. All samples contain phosphorylated AMP-activated protein kinase alpha (AMPKα) as a uniformly phosphorylated control for phosphoprotein extraction. Equal amounts of protein were loaded to each lane, as quantitated by tryptophan fluorescence spectrometry. β-Actin was used as a loading control. cCDCP1, cleaved CDCP1; CDCP1, CUB-domain containing protein 1; flCDCP1, full-length CDCP1; PKC, protein kinase C.
    Figure Legend Snippet: Western blot analysis of SFK_pY416− triple-negative breast cancer (TNBC) reveals that forkhead box protein 1 (FOXA1)+ samples tend to be SFK_pY527+ and FOXA1− samples tend to be SFK_pY527−. Paraffin sections of TNBC were extracted and analyzed by standard Western blot analysis technique. FOXA1, vimentin, and SFK_pY416 expression levels in tumor cells, as determined by immunohistochemistry (IHC), are summarized on top. A: All FOXA1+/SFK_pY416− samples tested were SFK_pY527+. The presence of SFK_pY416 in lanes 2, 4, and 6 is due to the presence of SFK_pY416+ lymphocytes in these tumor samples (shown in Supplemental Figure S1). B: Five of six FOXA1−/SFK_pY416− samples tested were SFK_pY527− (lanes 2, 3, and 5 to 7). Three FOXA1−/SFK_pY416+ tumor samples were run as controls. All samples contain phosphorylated AMP-activated protein kinase alpha (AMPKα) as a uniformly phosphorylated control for phosphoprotein extraction. Equal amounts of protein were loaded to each lane, as quantitated by tryptophan fluorescence spectrometry. β-Actin was used as a loading control. cCDCP1, cleaved CDCP1; CDCP1, CUB-domain containing protein 1; flCDCP1, full-length CDCP1; PKC, protein kinase C.

    Techniques Used: Western Blot, Expressing, Immunohistochemistry, Fluorescence

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    Structured Review

    Cell Signaling Technology Inc cdcp1 py734
    ( A ) MDCK cysts embedded within the collagen matrix were pretreated with NK4 (1 µg/ml), Torin1 (+, 50 nM; ++, 100 nM), rapamycin (+ Rapa, 50 nM; ++ Rapa, 100 nM), or dasatinib (20 nM, Das), and then incubated in the presence of HGF (50 ng/ml) for one day. Ki67 was visualized with an Alexa Fluor 488-conjugated antibody (green), and actin filaments were stained with Alexa Fluor 594-phalloidin (magenta). The arrowheads indicate transiently formed protrusions. ( B ) Diameter (µM) of cysts (n = 100). The dotted blue line indicates the average diameter of non-treated cysts. ( C ) Fraction of the total number of cysts counted (n > 100) with protrusions. ( D ) MDCK cysts embedded within the collagen matrix were incubated in the presence of HGF for the indicated time periods. Activated Src (pY418) was visualized with an Alexa Fluor 488-conjugated antibody (green), and actin filaments were stained with Alexa Fluor 594-phalloidin (magenta). The arrowheads indicate transiently formed protrusions. ( E ) mCherry-GPI-overexpressing MDCK cysts embedded within the collagen matrix were incubated in the presence of HGF for the indicated time periods. Actin filaments were stained with Alexa Fluor 488-phalloidin (green). ( F ) MDCK cysts embedded within the collagen matrix were incubated in the presence of HGF for the indicated time periods. The localization of <t>CDCP1</t> was visualized using an Alexa Fluor 488-conjugated antibody (green), and actin filaments were stained with Alexa Fluor 594-phalloidin (magenta). ( G ) Wild-type and CDCP1-knockout MDCK cysts were incubated in the presence of HGF for 1 day. Ki67 was visualized with an Alexa Fluor 594-conjugated antibody (magenta), and actin filaments were stained with Alexa Fluor 488-phalloidin. The arrowheads indicate transiently formed protrusions. The scale bars indicate 50 µm. ( H ) Diameter (µm) of cysts (n = 100). ( I ) Fraction of the total number of cysts counted (n > 100) with protrusions. The scale bars indicate 10 µm. The mean ratios ± SD were obtained from three independent experiments. **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; NS, not significantly different; ANOVA was calculated compared to HGF-treated cysts.
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    Images

    1) Product Images from "CUB domain-containing protein 1 controls HGF responses by integrating Src and Met–STAT3 signaling"

    Article Title: CUB domain-containing protein 1 controls HGF responses by integrating Src and Met–STAT3 signaling

    Journal: bioRxiv

    doi: 10.1101/789339

    ( A ) MDCK cysts embedded within the collagen matrix were pretreated with NK4 (1 µg/ml), Torin1 (+, 50 nM; ++, 100 nM), rapamycin (+ Rapa, 50 nM; ++ Rapa, 100 nM), or dasatinib (20 nM, Das), and then incubated in the presence of HGF (50 ng/ml) for one day. Ki67 was visualized with an Alexa Fluor 488-conjugated antibody (green), and actin filaments were stained with Alexa Fluor 594-phalloidin (magenta). The arrowheads indicate transiently formed protrusions. ( B ) Diameter (µM) of cysts (n = 100). The dotted blue line indicates the average diameter of non-treated cysts. ( C ) Fraction of the total number of cysts counted (n > 100) with protrusions. ( D ) MDCK cysts embedded within the collagen matrix were incubated in the presence of HGF for the indicated time periods. Activated Src (pY418) was visualized with an Alexa Fluor 488-conjugated antibody (green), and actin filaments were stained with Alexa Fluor 594-phalloidin (magenta). The arrowheads indicate transiently formed protrusions. ( E ) mCherry-GPI-overexpressing MDCK cysts embedded within the collagen matrix were incubated in the presence of HGF for the indicated time periods. Actin filaments were stained with Alexa Fluor 488-phalloidin (green). ( F ) MDCK cysts embedded within the collagen matrix were incubated in the presence of HGF for the indicated time periods. The localization of CDCP1 was visualized using an Alexa Fluor 488-conjugated antibody (green), and actin filaments were stained with Alexa Fluor 594-phalloidin (magenta). ( G ) Wild-type and CDCP1-knockout MDCK cysts were incubated in the presence of HGF for 1 day. Ki67 was visualized with an Alexa Fluor 594-conjugated antibody (magenta), and actin filaments were stained with Alexa Fluor 488-phalloidin. The arrowheads indicate transiently formed protrusions. The scale bars indicate 50 µm. ( H ) Diameter (µm) of cysts (n = 100). ( I ) Fraction of the total number of cysts counted (n > 100) with protrusions. The scale bars indicate 10 µm. The mean ratios ± SD were obtained from three independent experiments. **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; NS, not significantly different; ANOVA was calculated compared to HGF-treated cysts.
    Figure Legend Snippet: ( A ) MDCK cysts embedded within the collagen matrix were pretreated with NK4 (1 µg/ml), Torin1 (+, 50 nM; ++, 100 nM), rapamycin (+ Rapa, 50 nM; ++ Rapa, 100 nM), or dasatinib (20 nM, Das), and then incubated in the presence of HGF (50 ng/ml) for one day. Ki67 was visualized with an Alexa Fluor 488-conjugated antibody (green), and actin filaments were stained with Alexa Fluor 594-phalloidin (magenta). The arrowheads indicate transiently formed protrusions. ( B ) Diameter (µM) of cysts (n = 100). The dotted blue line indicates the average diameter of non-treated cysts. ( C ) Fraction of the total number of cysts counted (n > 100) with protrusions. ( D ) MDCK cysts embedded within the collagen matrix were incubated in the presence of HGF for the indicated time periods. Activated Src (pY418) was visualized with an Alexa Fluor 488-conjugated antibody (green), and actin filaments were stained with Alexa Fluor 594-phalloidin (magenta). The arrowheads indicate transiently formed protrusions. ( E ) mCherry-GPI-overexpressing MDCK cysts embedded within the collagen matrix were incubated in the presence of HGF for the indicated time periods. Actin filaments were stained with Alexa Fluor 488-phalloidin (green). ( F ) MDCK cysts embedded within the collagen matrix were incubated in the presence of HGF for the indicated time periods. The localization of CDCP1 was visualized using an Alexa Fluor 488-conjugated antibody (green), and actin filaments were stained with Alexa Fluor 594-phalloidin (magenta). ( G ) Wild-type and CDCP1-knockout MDCK cysts were incubated in the presence of HGF for 1 day. Ki67 was visualized with an Alexa Fluor 594-conjugated antibody (magenta), and actin filaments were stained with Alexa Fluor 488-phalloidin. The arrowheads indicate transiently formed protrusions. The scale bars indicate 50 µm. ( H ) Diameter (µm) of cysts (n = 100). ( I ) Fraction of the total number of cysts counted (n > 100) with protrusions. The scale bars indicate 10 µm. The mean ratios ± SD were obtained from three independent experiments. **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; NS, not significantly different; ANOVA was calculated compared to HGF-treated cysts.

    Techniques Used: Incubation, Staining, Knock-Out

    ( A ) Schematic illustration of the structure of CDCP1. In the cytoplasmic region, CDCP1 contains a Src-association motif around Tyr734 and two palmitoylation sites (Cys residues 689 and 690), which are required for CDCP1 localization in lipid rafts. Upon Tyr734 phosphorylation by Src, CDCP1 is additionally phosphorylated at Tyr762, resulting in direct association with PKCd, which promotes cell migration. In the extracellular region, CDCP1 contains three CUB domains that are required for protein–protein interactions. CDCP1 also harbors a proteolytic cleavage (shedding) site between the first and second CUB domains and can, thus, be present in either the full-length or cleaved form, depending on the cellular context. TM indicates the transmembrane domain. ( B ) Schematic diagram of CRISPR/Cas9-based generation of CDCP1-knockout MDCK cells. The blue text indicates the PAM sequence and the bold letters indicate the start codon. The red arrowhead indicates the cleavage site. ( C ) Immunoblotting analysis of CDCP1-knockout MDCK cells. Lysates from wild-type and CDCP1-knockout MDCK cells were subjected to immunoblotting using the indicated antibodies. ( D ) CDCP1-knockout MDCK cysts were incubated in the presence of HGF (50 ng/ml) for 4 or 6 days. Ki67 was visualized with an Alexa Fluor 488-conjugated antibody (green) and actin filaments were stained with Alexa Fluor 594-phalloidin (magenta). The scale bars indicate 10 µm.
    Figure Legend Snippet: ( A ) Schematic illustration of the structure of CDCP1. In the cytoplasmic region, CDCP1 contains a Src-association motif around Tyr734 and two palmitoylation sites (Cys residues 689 and 690), which are required for CDCP1 localization in lipid rafts. Upon Tyr734 phosphorylation by Src, CDCP1 is additionally phosphorylated at Tyr762, resulting in direct association with PKCd, which promotes cell migration. In the extracellular region, CDCP1 contains three CUB domains that are required for protein–protein interactions. CDCP1 also harbors a proteolytic cleavage (shedding) site between the first and second CUB domains and can, thus, be present in either the full-length or cleaved form, depending on the cellular context. TM indicates the transmembrane domain. ( B ) Schematic diagram of CRISPR/Cas9-based generation of CDCP1-knockout MDCK cells. The blue text indicates the PAM sequence and the bold letters indicate the start codon. The red arrowhead indicates the cleavage site. ( C ) Immunoblotting analysis of CDCP1-knockout MDCK cells. Lysates from wild-type and CDCP1-knockout MDCK cells were subjected to immunoblotting using the indicated antibodies. ( D ) CDCP1-knockout MDCK cysts were incubated in the presence of HGF (50 ng/ml) for 4 or 6 days. Ki67 was visualized with an Alexa Fluor 488-conjugated antibody (green) and actin filaments were stained with Alexa Fluor 594-phalloidin (magenta). The scale bars indicate 10 µm.

    Techniques Used: Migration, CRISPR, Knock-Out, Sequencing, Western Blot, Incubation, Staining

    ( A ) TRE–CDCP1–EGFP (CDCP1–EGFP cells)- and mutant (YF-EGFP or CG-EGFP)-harboring MDCK cells were incubated in the presence of Dox (1 µg/ml) for the indicated time periods. The cell lysates were subjected to immunoblotting using the indicated antibodies. ( B ) CDCP1–EGFP cells were incubated in the presence of Dox (1 µg/ml) for the indicated time periods. Actin filaments were stained with Alexa Fluor 594-phalloidin (magenta). The scale bars indicate 50 µm. ( C ) CDCP1–EGFP- and mutant-harboring MDCK cells were incubated in the presence of Dox (1 µg/ml) for 48 h. DRM and non-DRM fractions were separated on a sucrose-density gradient. Aliquots of the fractions were subjected to immunoblotting analysis, using the indicated antibodies. ( D ) DRM fractions of CDCP1–Myc-overexpressing MDCK cells were immunoprecipitated with an anti-Src or anti-Myc tag antibody. Immunoprecipitates were subjected to immunoblotting using the indicated antibodies (HC, heavy chain).
    Figure Legend Snippet: ( A ) TRE–CDCP1–EGFP (CDCP1–EGFP cells)- and mutant (YF-EGFP or CG-EGFP)-harboring MDCK cells were incubated in the presence of Dox (1 µg/ml) for the indicated time periods. The cell lysates were subjected to immunoblotting using the indicated antibodies. ( B ) CDCP1–EGFP cells were incubated in the presence of Dox (1 µg/ml) for the indicated time periods. Actin filaments were stained with Alexa Fluor 594-phalloidin (magenta). The scale bars indicate 50 µm. ( C ) CDCP1–EGFP- and mutant-harboring MDCK cells were incubated in the presence of Dox (1 µg/ml) for 48 h. DRM and non-DRM fractions were separated on a sucrose-density gradient. Aliquots of the fractions were subjected to immunoblotting analysis, using the indicated antibodies. ( D ) DRM fractions of CDCP1–Myc-overexpressing MDCK cells were immunoprecipitated with an anti-Src or anti-Myc tag antibody. Immunoprecipitates were subjected to immunoblotting using the indicated antibodies (HC, heavy chain).

    Techniques Used: Mutagenesis, Incubation, Western Blot, Staining, Immunoprecipitation

    ( A ) Schematic representation of the procedure used to analyze TRE–CDCP1–EGFP-harboring MDCK cysts (CDCP1–EGFP cysts). TRE–CDCP1–EGFP-harboring cells were cultured within a collagen matrix for 5 days to allow time for cyst formation, and then CDCP1–EGFP cysts were incubated in the presence of Dox for ∼4 days. ( B ) CDCP1–EGFP cysts embedded within the collagen matrix were incubated in the presence of Dox (1 µg/ml) for the indicated time periods. Actin filaments were stained with Alexa Fluor 594-phalloidin (magenta). Filled arrowheads indicate protruding cells and open arrowheads indicate multi-layered structures. ( C ) CDCP1– EGFP cysts were incubated in the presence of Dox for 2 days. Activated Src was visualized using an anti-Src pY418 antibody (magenta). ( D ) CDCP1–EGFP cysts embedded within the collagen matrix were pretreated with 20 nM dasatinib for 2 h, and then incubated with Dox for 4 days. ( E ) CDCP1–YF-EGFP and CDCP1–CG-EGFP cysts embedded within the collagen matrix were incubated in the presence of Dox for 4 days. Actin filaments were stained with Alexa Fluor 594-phalloidin (magenta). The scale bars indicate 50 µm. ( F ) Fraction of the total number of cysts counted (n > 150) with protrusions. The mean ratios ± SDs were determined from three independent experiments. ***, P < 0.001; ****, P < 0.0001; NS, not significantly different; ANOVA compared to the Dox-treated cysts.
    Figure Legend Snippet: ( A ) Schematic representation of the procedure used to analyze TRE–CDCP1–EGFP-harboring MDCK cysts (CDCP1–EGFP cysts). TRE–CDCP1–EGFP-harboring cells were cultured within a collagen matrix for 5 days to allow time for cyst formation, and then CDCP1–EGFP cysts were incubated in the presence of Dox for ∼4 days. ( B ) CDCP1–EGFP cysts embedded within the collagen matrix were incubated in the presence of Dox (1 µg/ml) for the indicated time periods. Actin filaments were stained with Alexa Fluor 594-phalloidin (magenta). Filled arrowheads indicate protruding cells and open arrowheads indicate multi-layered structures. ( C ) CDCP1– EGFP cysts were incubated in the presence of Dox for 2 days. Activated Src was visualized using an anti-Src pY418 antibody (magenta). ( D ) CDCP1–EGFP cysts embedded within the collagen matrix were pretreated with 20 nM dasatinib for 2 h, and then incubated with Dox for 4 days. ( E ) CDCP1–YF-EGFP and CDCP1–CG-EGFP cysts embedded within the collagen matrix were incubated in the presence of Dox for 4 days. Actin filaments were stained with Alexa Fluor 594-phalloidin (magenta). The scale bars indicate 50 µm. ( F ) Fraction of the total number of cysts counted (n > 150) with protrusions. The mean ratios ± SDs were determined from three independent experiments. ***, P < 0.001; ****, P < 0.0001; NS, not significantly different; ANOVA compared to the Dox-treated cysts.

    Techniques Used: Cell Culture, Incubation, Staining


    Figure Legend Snippet:

    Techniques Used:

    ( A ) GO analysis of CDCP1–EGFP cysts. Magenta bars indicate signaling related to GO annotations. ( B ) Regulatory networks upstream of STAT3 in CDCP1–EGFP and CDCP1–CG-EGFP cysts. Activation Z-scores are presented in Supplementary Table 2. Red and green symbols indicate transcript levels that were upregulated and downregulated by CDCP1 overexpression, respectively. Arrows are colored to indicate activation (orange), inhibition (blue), inconsistency with the state of the downstream molecule (yellow), and unknown effects (grey). ( C ) Heat map representing changes in the expression of STAT3 target genes in CDCP1–EGFP (WT) and CDCP1–CG-EGFP (CG) cysts.
    Figure Legend Snippet: ( A ) GO analysis of CDCP1–EGFP cysts. Magenta bars indicate signaling related to GO annotations. ( B ) Regulatory networks upstream of STAT3 in CDCP1–EGFP and CDCP1–CG-EGFP cysts. Activation Z-scores are presented in Supplementary Table 2. Red and green symbols indicate transcript levels that were upregulated and downregulated by CDCP1 overexpression, respectively. Arrows are colored to indicate activation (orange), inhibition (blue), inconsistency with the state of the downstream molecule (yellow), and unknown effects (grey). ( C ) Heat map representing changes in the expression of STAT3 target genes in CDCP1–EGFP (WT) and CDCP1–CG-EGFP (CG) cysts.

    Techniques Used: Activation Assay, Over Expression, Inhibition, Expressing

    ( A ) MDCK cells overexpressing wild-type or mutant CDCP1 (Myc-tagged variants) were embedded within the collagen matrix and cultured for 9 days. Cyst lysates were subjected to immunoblotting using the indicated antibodies. ( B ) CDCP1–EGFP cysts were incubated with Dox (1 µg/ml) for 2 or 4 days, and then subjected to quantitative real-time PCR. Relative mRNA-expression levels were calculated by setting the mean value for non-treated cysts to 1. The mean ratios ± SDs were determined from three independent experiments. ANOVA calculations were performed to compare the results with those from non-treated cysts. ( C ) CDCP1–EGFP cysts were pretreated with the indicated STAT3-specific inhibitors for 2 h and then incubated with Dox (1 µg/ml) for 4 days.. ( D ) STAT3–Y705F-overexpressing CDCP1–EGFP cysts were incubated with Dox (1 µg/ml) for 4 days. Actin filaments were stained with Alexa Fluor 594-phalloidin (magenta). The scale bars indicate 50 µm. ( E, F ) MDCK cysts embedded within the collagen matrix were pretreated with S3i-201 (+ S3i, 50 nM; ++ S3i, 100 nM) or Stattic (+ Sta, 2.5 nM; ++ Sta, 5.0 nM) for 2 h and then incubated with HGF (50 ng/ml) for 1 day. ( E ) Diameter (µm) of cysts (n = 100). ( F ) Fraction of the total number of cysts counted (n > 100) with protrusions. The mean ratios ± SDs were obtained from three independent experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; NS, not significantly different; ANOVA was calculated compared to the HGF-treated cysts.
    Figure Legend Snippet: ( A ) MDCK cells overexpressing wild-type or mutant CDCP1 (Myc-tagged variants) were embedded within the collagen matrix and cultured for 9 days. Cyst lysates were subjected to immunoblotting using the indicated antibodies. ( B ) CDCP1–EGFP cysts were incubated with Dox (1 µg/ml) for 2 or 4 days, and then subjected to quantitative real-time PCR. Relative mRNA-expression levels were calculated by setting the mean value for non-treated cysts to 1. The mean ratios ± SDs were determined from three independent experiments. ANOVA calculations were performed to compare the results with those from non-treated cysts. ( C ) CDCP1–EGFP cysts were pretreated with the indicated STAT3-specific inhibitors for 2 h and then incubated with Dox (1 µg/ml) for 4 days.. ( D ) STAT3–Y705F-overexpressing CDCP1–EGFP cysts were incubated with Dox (1 µg/ml) for 4 days. Actin filaments were stained with Alexa Fluor 594-phalloidin (magenta). The scale bars indicate 50 µm. ( E, F ) MDCK cysts embedded within the collagen matrix were pretreated with S3i-201 (+ S3i, 50 nM; ++ S3i, 100 nM) or Stattic (+ Sta, 2.5 nM; ++ Sta, 5.0 nM) for 2 h and then incubated with HGF (50 ng/ml) for 1 day. ( E ) Diameter (µm) of cysts (n = 100). ( F ) Fraction of the total number of cysts counted (n > 100) with protrusions. The mean ratios ± SDs were obtained from three independent experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; NS, not significantly different; ANOVA was calculated compared to the HGF-treated cysts.

    Techniques Used: Mutagenesis, Cell Culture, Western Blot, Incubation, Real-time Polymerase Chain Reaction, Expressing, Staining

    ( A ) CDCP1–EGFP cysts embedded within the collagen matrix were incubated with Dox (1 µg/ml) for the indicated time periods. The basement membrane was visualized with an anti-laminin antibody (magenta). The arrowheads indicate protruding cells, and the open arrowheads indicate multi-layered structures. ( B ) CDCP1–EGFP cysts were pretreated with 20 µM marimastat for 2 h and then incubated with Dox (1 µg/ml) for 4 days. The basement membrane was visualized using an anti-laminin antibody (magenta). ( C ) The histogram depicting the percentage of cells with protrusions was calculated by setting the total number of cysts to 100% (n > 150). ( D, E ) MDCK cysts embedded within the collagen matrix were pretreated with marimastat (+ Mari, 10 µM; ++ Mari, 20 µM) for 2 h and then incubated in the presence of HGF (50 ng/ml) for 1 day. ( D ) Diameter (µm) of cysts (n = 100). The dotted blue line indicates the average diameter of non-treated cysts. ( E ) Fraction of the total number of cysts counted (n > 100). The mean ratios ± SDs were obtained from three independent experiments. *, P < 0.05; **, P < 0.01; **** P < 0.0001. Two-way ANOVA was performed relative to the HGF-treated cysts. ( F ) TRE–CDCP1–mCherry-harboring MDCK cells were embedded within a 2% DQ-collagen-containing matrix. CDCP1–mCherry cysts were pretreated with 20 µM marimastat for 2 h and then incubated with Dox (1 µg/ml) for 4 days. DQ fluorescence (green) was visualized under a fluorescent microscope. Scale bars indicate 50 µm. ( G ) STAT3–MER-overexpressing cysts embedded within the collagen matrix were incubated with 1 µM 4-OHT for 4 days. STAT3–MER was visualized with ERα antibody (green) and actin filaments were stained with Alexa Fluor 594-phalloidin (magenta). The scale bars indicate 50 µm.
    Figure Legend Snippet: ( A ) CDCP1–EGFP cysts embedded within the collagen matrix were incubated with Dox (1 µg/ml) for the indicated time periods. The basement membrane was visualized with an anti-laminin antibody (magenta). The arrowheads indicate protruding cells, and the open arrowheads indicate multi-layered structures. ( B ) CDCP1–EGFP cysts were pretreated with 20 µM marimastat for 2 h and then incubated with Dox (1 µg/ml) for 4 days. The basement membrane was visualized using an anti-laminin antibody (magenta). ( C ) The histogram depicting the percentage of cells with protrusions was calculated by setting the total number of cysts to 100% (n > 150). ( D, E ) MDCK cysts embedded within the collagen matrix were pretreated with marimastat (+ Mari, 10 µM; ++ Mari, 20 µM) for 2 h and then incubated in the presence of HGF (50 ng/ml) for 1 day. ( D ) Diameter (µm) of cysts (n = 100). The dotted blue line indicates the average diameter of non-treated cysts. ( E ) Fraction of the total number of cysts counted (n > 100). The mean ratios ± SDs were obtained from three independent experiments. *, P < 0.05; **, P < 0.01; **** P < 0.0001. Two-way ANOVA was performed relative to the HGF-treated cysts. ( F ) TRE–CDCP1–mCherry-harboring MDCK cells were embedded within a 2% DQ-collagen-containing matrix. CDCP1–mCherry cysts were pretreated with 20 µM marimastat for 2 h and then incubated with Dox (1 µg/ml) for 4 days. DQ fluorescence (green) was visualized under a fluorescent microscope. Scale bars indicate 50 µm. ( G ) STAT3–MER-overexpressing cysts embedded within the collagen matrix were incubated with 1 µM 4-OHT for 4 days. STAT3–MER was visualized with ERα antibody (green) and actin filaments were stained with Alexa Fluor 594-phalloidin (magenta). The scale bars indicate 50 µm.

    Techniques Used: Incubation, Fluorescence, Microscopy, Staining

    ( A ) CDCP1–EGFP cysts were pretreated with the indicated Met-specific inhibitors for 2 h and then incubated with Dox (1 µg/ml) for 4 days. ( B ) Schematic representation of CDCP1 and the deletion mutants. SP, signal peptide; CUB, CUB domain; TM, transmembrane domain; CD, cytosolic domain. ( C ) Lysates from HEK293 cells overexpressing both CDCP1-Myc and Met were subjected to immunoprecipitation with an anti-Myc tag antibody. Immunoprecipitates were subjected to immunoblotting using the indicated antibodies (IgG HC, IgG heavy chain). ( D ) CDCP1-myc- and CDCP1-PR-myc-overexpressing MDCK cells were embedded within collagen matrix and cultured for 9 days. Cyst lysates were subjected to immunoblotting using the indicated antibodies. ( E ) CDCP1–PR–EGFP cysts embedded within the collagen matrix were incubated with Dox for 4 days. Actin filaments were stained with Alexa Fluor 594-phalloidin (magenta). The scale bars indicate 50 µm. ( F ) Fraction of the total number of cysts counted (n > 150) with protrusions. The mean ratios ± SDs were obtained from three independent experiments. **, P < 0.001; ****, P < 0.0001; NS, not significantly different; ANOVA was calculated compared to the Dox-treated cysts. ( G ) Schematic model of the role of CDCP1–Met association in Src-induced STAT3 phosphorylation.
    Figure Legend Snippet: ( A ) CDCP1–EGFP cysts were pretreated with the indicated Met-specific inhibitors for 2 h and then incubated with Dox (1 µg/ml) for 4 days. ( B ) Schematic representation of CDCP1 and the deletion mutants. SP, signal peptide; CUB, CUB domain; TM, transmembrane domain; CD, cytosolic domain. ( C ) Lysates from HEK293 cells overexpressing both CDCP1-Myc and Met were subjected to immunoprecipitation with an anti-Myc tag antibody. Immunoprecipitates were subjected to immunoblotting using the indicated antibodies (IgG HC, IgG heavy chain). ( D ) CDCP1-myc- and CDCP1-PR-myc-overexpressing MDCK cells were embedded within collagen matrix and cultured for 9 days. Cyst lysates were subjected to immunoblotting using the indicated antibodies. ( E ) CDCP1–PR–EGFP cysts embedded within the collagen matrix were incubated with Dox for 4 days. Actin filaments were stained with Alexa Fluor 594-phalloidin (magenta). The scale bars indicate 50 µm. ( F ) Fraction of the total number of cysts counted (n > 150) with protrusions. The mean ratios ± SDs were obtained from three independent experiments. **, P < 0.001; ****, P < 0.0001; NS, not significantly different; ANOVA was calculated compared to the Dox-treated cysts. ( G ) Schematic model of the role of CDCP1–Met association in Src-induced STAT3 phosphorylation.

    Techniques Used: Incubation, Immunoprecipitation, Western Blot, Cell Culture, Staining

    ( A ) Wild-type and CDCP1-knockout TRE–Met-harboring MDCK cells embedded within the collagen matrix were incubated with HGF (50 ng/ml) or Dox (1 µg/ml) for 4 days. Met was visualized with an anti-Met antibody (green). Scale bars indicate 50 µm. ( B ) A schematic model of the role of CDCP1–Src in HGF-induced invasive growth. CDCP1–Src activates Met–STAT3 signaling in lipid rafts, leading to invasive growth by inducing ECM rearrangement and cell growth/proliferation. The mTOR pathway also contributes to cell growth.
    Figure Legend Snippet: ( A ) Wild-type and CDCP1-knockout TRE–Met-harboring MDCK cells embedded within the collagen matrix were incubated with HGF (50 ng/ml) or Dox (1 µg/ml) for 4 days. Met was visualized with an anti-Met antibody (green). Scale bars indicate 50 µm. ( B ) A schematic model of the role of CDCP1–Src in HGF-induced invasive growth. CDCP1–Src activates Met–STAT3 signaling in lipid rafts, leading to invasive growth by inducing ECM rearrangement and cell growth/proliferation. The mTOR pathway also contributes to cell growth.

    Techniques Used: Knock-Out, Incubation

    ( A ) Total lysates from MCF7, T47D, and MDA-MB231 cells were subjected to immunoblotting using the indicated antibodies. ( B ) MDA-MB231 cells treated with or without HGF (100 ng/ml) were transfected with the indicated CDCP1 siRNAs, and total cell lysates were subjected to immunoblotting using the indicated antibodies. ( C ) The in vitro migration activities of MDA-MB231 cells treated with control and siRNAs were examined by performing transwell assays in the presence or absence of HGF. ( D ) The in vitro invasion activity of control- and siRNA-treated MDA-MB231 cells was examined by performing Matrigel transwell assays in the presence or absence of HGF. Rescue experiments were also performed by re-expressing wild-type CDCP1-myc, CDCP1-CG-myc, and CDCP1-PR-myc. ( E, F ) Formation of lamellipodia in control- and siRNA-treated MDA-MB231 cells was examined by immunofluorescent analysis for F-actin in the presence or absence of HGF (left panels). Yellow arrowheads indicate lamellipodia. Rescue experiments were also performed by re-expressing wild-type CDCP1-myc, CDCP1-CG-myc, and CDCP1-PR-myc (upper right panels). The ratios of the length of lamellipodial membrane to the total cell perimeter were calculated from at least 30 cells of each cell type (lower right graph). The scale bars indicate 50 µm. The mean ratios ± SDs were obtained from three independent experiments. *, P < 0.05; **, P < 0.001; ***, P < 0.001; ****, P < 0.0001; NS, not significantly different; two-way ANOVA.
    Figure Legend Snippet: ( A ) Total lysates from MCF7, T47D, and MDA-MB231 cells were subjected to immunoblotting using the indicated antibodies. ( B ) MDA-MB231 cells treated with or without HGF (100 ng/ml) were transfected with the indicated CDCP1 siRNAs, and total cell lysates were subjected to immunoblotting using the indicated antibodies. ( C ) The in vitro migration activities of MDA-MB231 cells treated with control and siRNAs were examined by performing transwell assays in the presence or absence of HGF. ( D ) The in vitro invasion activity of control- and siRNA-treated MDA-MB231 cells was examined by performing Matrigel transwell assays in the presence or absence of HGF. Rescue experiments were also performed by re-expressing wild-type CDCP1-myc, CDCP1-CG-myc, and CDCP1-PR-myc. ( E, F ) Formation of lamellipodia in control- and siRNA-treated MDA-MB231 cells was examined by immunofluorescent analysis for F-actin in the presence or absence of HGF (left panels). Yellow arrowheads indicate lamellipodia. Rescue experiments were also performed by re-expressing wild-type CDCP1-myc, CDCP1-CG-myc, and CDCP1-PR-myc (upper right panels). The ratios of the length of lamellipodial membrane to the total cell perimeter were calculated from at least 30 cells of each cell type (lower right graph). The scale bars indicate 50 µm. The mean ratios ± SDs were obtained from three independent experiments. *, P < 0.05; **, P < 0.001; ***, P < 0.001; ****, P < 0.0001; NS, not significantly different; two-way ANOVA.

    Techniques Used: Western Blot, Transfection, In Vitro, Migration, Activity Assay, Expressing

    ( A ) Schematic diagram of CRISPR/Cas9-based generation of Cdcp1 -knockout mouse. The blue text indicates the PAM sequence and the bold letters indicate the start codon. The red arrowhead indicates the cleavage site. ( B ) PCR verification of deletion of the 1st exon of Cdcp1 using the forward and reverse primers depicted in panel ( A ). ( C ) Representative picture of wild-type (+/+), Cdcp1 heterozygous (+/–), and homozygous knockout (–/–) littermate mice at 8 weeks of age. ( D, E ) Whole body ( D ) and left kidney ( E ) weight of wild-type (+/+) and Cdcp1 homozygous knockout (–/–) mice at 8 weeks of age. The mean ratios ± SDs were obtained from ten mice per group. NS, not significantly different; unpaired two-tailed t -test
    Figure Legend Snippet: ( A ) Schematic diagram of CRISPR/Cas9-based generation of Cdcp1 -knockout mouse. The blue text indicates the PAM sequence and the bold letters indicate the start codon. The red arrowhead indicates the cleavage site. ( B ) PCR verification of deletion of the 1st exon of Cdcp1 using the forward and reverse primers depicted in panel ( A ). ( C ) Representative picture of wild-type (+/+), Cdcp1 heterozygous (+/–), and homozygous knockout (–/–) littermate mice at 8 weeks of age. ( D, E ) Whole body ( D ) and left kidney ( E ) weight of wild-type (+/+) and Cdcp1 homozygous knockout (–/–) mice at 8 weeks of age. The mean ratios ± SDs were obtained from ten mice per group. NS, not significantly different; unpaired two-tailed t -test

    Techniques Used: CRISPR, Knock-Out, Sequencing, Two Tailed Test

    ( A ) Cdcp1 wild-type (+/+), heterozygous (+/–), and homozygous knockout (–/–) mice at 8 weeks of age were subjected to left UNX or a sham operation. Regenerative growth of the remaining left kidney was analyzed 8 weeks after operation and increases in remaining kidney/body weight ratios were assessed. ( B ) The mean ratios ± SDs were obtained from 10 mice per group. ( C ) The remaining kidney was removed from UNX- or sham-operated mice, and proximal tubules were stained with FITC-LTL (green). The scale bars indicate 50 µm. ( D ) The proximal tubule thickness was determined by the length of FITC-LTL-stained epithelial cell layer (n = 100). The mean ratios ± SDs were obtained from 5 mice per group. *, P < 0.05; **, P < 0.01; ****, P < 0.0001; NS, not significantly different; two-way ANOVA.
    Figure Legend Snippet: ( A ) Cdcp1 wild-type (+/+), heterozygous (+/–), and homozygous knockout (–/–) mice at 8 weeks of age were subjected to left UNX or a sham operation. Regenerative growth of the remaining left kidney was analyzed 8 weeks after operation and increases in remaining kidney/body weight ratios were assessed. ( B ) The mean ratios ± SDs were obtained from 10 mice per group. ( C ) The remaining kidney was removed from UNX- or sham-operated mice, and proximal tubules were stained with FITC-LTL (green). The scale bars indicate 50 µm. ( D ) The proximal tubule thickness was determined by the length of FITC-LTL-stained epithelial cell layer (n = 100). The mean ratios ± SDs were obtained from 5 mice per group. *, P < 0.05; **, P < 0.01; ****, P < 0.0001; NS, not significantly different; two-way ANOVA.

    Techniques Used: Knock-Out, Staining

    ( A ) Cdcp1 wild-type (+/+) and homozygous knockout (–/–) mice at 8 weeks of age were subjected to left UNX or a sham operation. Compensatory renal growth of the remaining left kidney was analyzed at the indicated time points after operation and increases in remaining kidney/body weight ratios were assessed. The mean ratios ± SDs were obtained from at least 6 mice per group. ( B–D, F, G ) The remaining kidney after UNX was subjected to microscopic immunofluorescence analysis with specific antibodies against Met pY1234-1235 ( B ), STAT3 pY705 ( C ), Ki67 ( D ), collagen IV ( F ), MMP9 ( G ), and Alexa Fluor 594-conjugated secondary antibody (magenta). Proximal tubules were visualized by staining with FITC-LTL (green). The scale bars indicate 50 µm. Ki67-positive proximal tubules (%) were estimated by calculating the ratio of Ki67-stained tubules to the total number of tubules (n > 100) ( E ). The mean ratios ± SD were obtained from 3 mice per group. *, P < 0.05; **, P < 0.01; ****, P < 0.0001; NS, not significantly different; two-way ANOVA was calculated compared to sham operated control. ( H ) Schematic model of HGF-induced adaptive renal regeneration. CDCP1–Src regulated Met–STAT3 signaling leading to compensatory renal growth through the induction of ECM rearrangement and cell growth/proliferation.
    Figure Legend Snippet: ( A ) Cdcp1 wild-type (+/+) and homozygous knockout (–/–) mice at 8 weeks of age were subjected to left UNX or a sham operation. Compensatory renal growth of the remaining left kidney was analyzed at the indicated time points after operation and increases in remaining kidney/body weight ratios were assessed. The mean ratios ± SDs were obtained from at least 6 mice per group. ( B–D, F, G ) The remaining kidney after UNX was subjected to microscopic immunofluorescence analysis with specific antibodies against Met pY1234-1235 ( B ), STAT3 pY705 ( C ), Ki67 ( D ), collagen IV ( F ), MMP9 ( G ), and Alexa Fluor 594-conjugated secondary antibody (magenta). Proximal tubules were visualized by staining with FITC-LTL (green). The scale bars indicate 50 µm. Ki67-positive proximal tubules (%) were estimated by calculating the ratio of Ki67-stained tubules to the total number of tubules (n > 100) ( E ). The mean ratios ± SD were obtained from 3 mice per group. *, P < 0.05; **, P < 0.01; ****, P < 0.0001; NS, not significantly different; two-way ANOVA was calculated compared to sham operated control. ( H ) Schematic model of HGF-induced adaptive renal regeneration. CDCP1–Src regulated Met–STAT3 signaling leading to compensatory renal growth through the induction of ECM rearrangement and cell growth/proliferation.

    Techniques Used: Knock-Out, Immunofluorescence, Staining

    ( A ) The remaining kidneys of wild-type (+/+) and Cdcp1- homozygous knockout (–/–) mice after UNX were subjected to immunofluorescence microscopy analysis using a primary antibody against CDCP1 pY734 and an Alexa Fluor 594-conjugated secondary antibody (magenta). ( B–D ) The remaining kidneys of wild-type ( Cdcp1 +/+) mice after UNX were subjected to immunofluorescence microscopy analysis using specific primary antibodies against Met pY1234-1235 ( B ), STAT3 pY705 ( C ), and Src pY418 ( D ) and an Alexa Fluor 488-conjugated secondary antibody (green). The endosome marker EEA1 was visualized using an Alexa Fluor 594-conjugated secondary antibody (magenta). ( E ) The remaining kidneys of Cdcp1 wild-type (+/+) and Cdcp1 -homozygous knockout (–/–) mice after UNX were subjected to immunofluorescence microscopy analysis using a primary antibody against MMP2 and an Alexa Fluor 594-conjugated secondary antibody (magenta). Proximal tubules were visualized by staining with FITC-LTL (green). The scale bars indicate 50 µm. ( F ) Schematic diagram summarizing differences in cellular events observed during compensatory renal growth of wild-type (+/+) and Cdcp1 -homozygous knockout (–/–) mice.
    Figure Legend Snippet: ( A ) The remaining kidneys of wild-type (+/+) and Cdcp1- homozygous knockout (–/–) mice after UNX were subjected to immunofluorescence microscopy analysis using a primary antibody against CDCP1 pY734 and an Alexa Fluor 594-conjugated secondary antibody (magenta). ( B–D ) The remaining kidneys of wild-type ( Cdcp1 +/+) mice after UNX were subjected to immunofluorescence microscopy analysis using specific primary antibodies against Met pY1234-1235 ( B ), STAT3 pY705 ( C ), and Src pY418 ( D ) and an Alexa Fluor 488-conjugated secondary antibody (green). The endosome marker EEA1 was visualized using an Alexa Fluor 594-conjugated secondary antibody (magenta). ( E ) The remaining kidneys of Cdcp1 wild-type (+/+) and Cdcp1 -homozygous knockout (–/–) mice after UNX were subjected to immunofluorescence microscopy analysis using a primary antibody against MMP2 and an Alexa Fluor 594-conjugated secondary antibody (magenta). Proximal tubules were visualized by staining with FITC-LTL (green). The scale bars indicate 50 µm. ( F ) Schematic diagram summarizing differences in cellular events observed during compensatory renal growth of wild-type (+/+) and Cdcp1 -homozygous knockout (–/–) mice.

    Techniques Used: Knock-Out, Immunofluorescence, Microscopy, Marker, Staining

    ( A ) CDCP1 downregulation induced the formation of a luminal structure. CDCP1–EGFP cysts embedded within the collagen matrix were incubated in the presence of Dox (1 µg/ml) for 4 days. Cysts were then incubated for an additional 5 days in the absence of Dox. ( B ) CDCP1 upregulation induced Cytokeratin 14 (CK14) expression. TRE–CDCP1–EGFP-harboring cysts were incubated with Dox (1 µg/ml) for 4 days. CK14 was visualized with a specific antibody (magenta). The scale bars indicate 50 µm.
    Figure Legend Snippet: ( A ) CDCP1 downregulation induced the formation of a luminal structure. CDCP1–EGFP cysts embedded within the collagen matrix were incubated in the presence of Dox (1 µg/ml) for 4 days. Cysts were then incubated for an additional 5 days in the absence of Dox. ( B ) CDCP1 upregulation induced Cytokeratin 14 (CK14) expression. TRE–CDCP1–EGFP-harboring cysts were incubated with Dox (1 µg/ml) for 4 days. CK14 was visualized with a specific antibody (magenta). The scale bars indicate 50 µm.

    Techniques Used: Incubation, Expressing

    ( A, B ) Correlations of CDCP1- and Met-expression levels with the prognoses of patients with breast cancer ( A ) or kidney clear cell carcinoma ( B ) were estimated using the Kaplan–Meier method, based on the transcriptome dataset from the TCGA project. Statistical significance was calculated by performing a log-rank test.
    Figure Legend Snippet: ( A, B ) Correlations of CDCP1- and Met-expression levels with the prognoses of patients with breast cancer ( A ) or kidney clear cell carcinoma ( B ) were estimated using the Kaplan–Meier method, based on the transcriptome dataset from the TCGA project. Statistical significance was calculated by performing a log-rank test.

    Techniques Used: Expressing

    p cdcp1 py734  (Cell Signaling Technology Inc)


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    Structured Review

    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.
    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

    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.
    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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  • 93
    Cell Signaling Technology Inc anti cdcp1 py734
    (A, B, C, D) MDCK cysts embedded within the collagen matrix were pretreated with NK4 (1 μg/ml), Torin1 (+, 50 nM; ++, 100 nM), rapamycin (+ Rapa, 50 nM; ++ Rapa, 100 nM), dasatinib (20 nM, Dasa), saracatinib (10 μM, Sara), or simvastatin (2 μM, Statin) and then incubated in the presence of HGF (50 ng/ml) for 1 d. (A) Ki67 was visualized with an Alexa Fluor 488–conjugated antibody (green), and actin filaments were stained with Alexa Fluor 594–phalloidin (magenta); arrowheads indicate transiently formed protrusions. Diameter (μm) of cysts (n = 100). (B) Dotted blue line indicates the average diameter of non-treated cysts. (C) Ratio of Ki67-positive cell in cyst (n = 50). (D) Fraction of the total number of cysts counted (n > 100) with protrusions. (E) MDCK cysts embedded within the collagen matrix were incubated in the presence of HGF (50 ng/ml) for the indicated time periods. Activated Src (pY418) was visualized with an Alexa Fluor 488–conjugated antibody (green), and actin filaments were stained with Alexa Fluor 594-phalloidin (magenta); arrowheads indicate transiently formed protrusions. (F) mCherry-GPI–overexpressing MDCK cysts embedded within the collagen matrix were incubated in the presence of HGF (50 ng/ml) for the indicated time periods. Actin filaments were stained with Alexa Fluor 488-phalloidin (green); arrowheads indicate transiently formed protrusions. (G) MDCK cysts embedded within the collagen matrix were incubated in the presence of HGF (50 ng/ml) for the indicated time periods. Localization of <t>CDCP1</t> was visualized using an Alexa Fluor 488–conjugated antibody (green), and actin filaments were stained with Alexa Fluor 594-phalloidin (magenta); arrowheads indicate transiently formed protrusions. (H, I, J) Wild-type and CDCP1 -knockout MDCK cysts were incubated in the presence of HGF (50 ng/ml) for 1 d. (H) Ki67 was visualized with an Alexa Fluor 488-conjugated antibody (green), and actin filaments were stained with Alexa Fluor 594-phalloidin (magenta); arrowheads indicate transiently formed protrusions. (I) Diameter (μm) of cysts (n = 100). (J) Fraction of the total number of cysts counted (n > 100) with protrusions. Scale bars: 10 μm. Data information: In (B, C, D, I, J), the mean ratios ± SD were obtained from three independent experiments. * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001; NS, not significantly different; two-way ANOVA was calculated compared with HGF-treated cysts.
    Anti Cdcp1 Py734, 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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    86
    Cell Signaling Technology Inc anti cdcp1 py734 antibody
    (A, B, C, D) MDCK cysts embedded within the collagen matrix were pretreated with NK4 (1 μg/ml), Torin1 (+, 50 nM; ++, 100 nM), rapamycin (+ Rapa, 50 nM; ++ Rapa, 100 nM), dasatinib (20 nM, Dasa), saracatinib (10 μM, Sara), or simvastatin (2 μM, Statin) and then incubated in the presence of HGF (50 ng/ml) for 1 d. (A) Ki67 was visualized with an Alexa Fluor 488–conjugated antibody (green), and actin filaments were stained with Alexa Fluor 594–phalloidin (magenta); arrowheads indicate transiently formed protrusions. Diameter (μm) of cysts (n = 100). (B) Dotted blue line indicates the average diameter of non-treated cysts. (C) Ratio of Ki67-positive cell in cyst (n = 50). (D) Fraction of the total number of cysts counted (n > 100) with protrusions. (E) MDCK cysts embedded within the collagen matrix were incubated in the presence of HGF (50 ng/ml) for the indicated time periods. Activated Src (pY418) was visualized with an Alexa Fluor 488–conjugated antibody (green), and actin filaments were stained with Alexa Fluor 594-phalloidin (magenta); arrowheads indicate transiently formed protrusions. (F) mCherry-GPI–overexpressing MDCK cysts embedded within the collagen matrix were incubated in the presence of HGF (50 ng/ml) for the indicated time periods. Actin filaments were stained with Alexa Fluor 488-phalloidin (green); arrowheads indicate transiently formed protrusions. (G) MDCK cysts embedded within the collagen matrix were incubated in the presence of HGF (50 ng/ml) for the indicated time periods. Localization of <t>CDCP1</t> was visualized using an Alexa Fluor 488–conjugated antibody (green), and actin filaments were stained with Alexa Fluor 594-phalloidin (magenta); arrowheads indicate transiently formed protrusions. (H, I, J) Wild-type and CDCP1 -knockout MDCK cysts were incubated in the presence of HGF (50 ng/ml) for 1 d. (H) Ki67 was visualized with an Alexa Fluor 488-conjugated antibody (green), and actin filaments were stained with Alexa Fluor 594-phalloidin (magenta); arrowheads indicate transiently formed protrusions. (I) Diameter (μm) of cysts (n = 100). (J) Fraction of the total number of cysts counted (n > 100) with protrusions. Scale bars: 10 μm. Data information: In (B, C, D, I, J), the mean ratios ± SD were obtained from three independent experiments. * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001; NS, not significantly different; two-way ANOVA was calculated compared with HGF-treated cysts.
    Anti Cdcp1 Py734 Antibody, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/anti cdcp1 py734 antibody/product/Cell Signaling Technology Inc
    Average 86 stars, based on 1 article reviews
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    93
    Cell Signaling Technology Inc cdcp1 py734
    Antibodies and Dilutions Used for IHC and Western Blot Analysis
    Cdcp1 Py734, 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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    95
    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.
    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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    Image Search Results


    (A, B, C, D) MDCK cysts embedded within the collagen matrix were pretreated with NK4 (1 μg/ml), Torin1 (+, 50 nM; ++, 100 nM), rapamycin (+ Rapa, 50 nM; ++ Rapa, 100 nM), dasatinib (20 nM, Dasa), saracatinib (10 μM, Sara), or simvastatin (2 μM, Statin) and then incubated in the presence of HGF (50 ng/ml) for 1 d. (A) Ki67 was visualized with an Alexa Fluor 488–conjugated antibody (green), and actin filaments were stained with Alexa Fluor 594–phalloidin (magenta); arrowheads indicate transiently formed protrusions. Diameter (μm) of cysts (n = 100). (B) Dotted blue line indicates the average diameter of non-treated cysts. (C) Ratio of Ki67-positive cell in cyst (n = 50). (D) Fraction of the total number of cysts counted (n > 100) with protrusions. (E) MDCK cysts embedded within the collagen matrix were incubated in the presence of HGF (50 ng/ml) for the indicated time periods. Activated Src (pY418) was visualized with an Alexa Fluor 488–conjugated antibody (green), and actin filaments were stained with Alexa Fluor 594-phalloidin (magenta); arrowheads indicate transiently formed protrusions. (F) mCherry-GPI–overexpressing MDCK cysts embedded within the collagen matrix were incubated in the presence of HGF (50 ng/ml) for the indicated time periods. Actin filaments were stained with Alexa Fluor 488-phalloidin (green); arrowheads indicate transiently formed protrusions. (G) MDCK cysts embedded within the collagen matrix were incubated in the presence of HGF (50 ng/ml) for the indicated time periods. Localization of CDCP1 was visualized using an Alexa Fluor 488–conjugated antibody (green), and actin filaments were stained with Alexa Fluor 594-phalloidin (magenta); arrowheads indicate transiently formed protrusions. (H, I, J) Wild-type and CDCP1 -knockout MDCK cysts were incubated in the presence of HGF (50 ng/ml) for 1 d. (H) Ki67 was visualized with an Alexa Fluor 488-conjugated antibody (green), and actin filaments were stained with Alexa Fluor 594-phalloidin (magenta); arrowheads indicate transiently formed protrusions. (I) Diameter (μm) of cysts (n = 100). (J) Fraction of the total number of cysts counted (n > 100) with protrusions. Scale bars: 10 μm. Data information: In (B, C, D, I, J), the mean ratios ± SD were obtained from three independent experiments. * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001; NS, not significantly different; two-way ANOVA was calculated compared with HGF-treated cysts.

    Journal: Life Science Alliance

    Article Title: CDCP1 promotes compensatory renal growth by integrating Src and Met signaling

    doi: 10.26508/lsa.202000832

    Figure Lengend Snippet: (A, B, C, D) MDCK cysts embedded within the collagen matrix were pretreated with NK4 (1 μg/ml), Torin1 (+, 50 nM; ++, 100 nM), rapamycin (+ Rapa, 50 nM; ++ Rapa, 100 nM), dasatinib (20 nM, Dasa), saracatinib (10 μM, Sara), or simvastatin (2 μM, Statin) and then incubated in the presence of HGF (50 ng/ml) for 1 d. (A) Ki67 was visualized with an Alexa Fluor 488–conjugated antibody (green), and actin filaments were stained with Alexa Fluor 594–phalloidin (magenta); arrowheads indicate transiently formed protrusions. Diameter (μm) of cysts (n = 100). (B) Dotted blue line indicates the average diameter of non-treated cysts. (C) Ratio of Ki67-positive cell in cyst (n = 50). (D) Fraction of the total number of cysts counted (n > 100) with protrusions. (E) MDCK cysts embedded within the collagen matrix were incubated in the presence of HGF (50 ng/ml) for the indicated time periods. Activated Src (pY418) was visualized with an Alexa Fluor 488–conjugated antibody (green), and actin filaments were stained with Alexa Fluor 594-phalloidin (magenta); arrowheads indicate transiently formed protrusions. (F) mCherry-GPI–overexpressing MDCK cysts embedded within the collagen matrix were incubated in the presence of HGF (50 ng/ml) for the indicated time periods. Actin filaments were stained with Alexa Fluor 488-phalloidin (green); arrowheads indicate transiently formed protrusions. (G) MDCK cysts embedded within the collagen matrix were incubated in the presence of HGF (50 ng/ml) for the indicated time periods. Localization of CDCP1 was visualized using an Alexa Fluor 488–conjugated antibody (green), and actin filaments were stained with Alexa Fluor 594-phalloidin (magenta); arrowheads indicate transiently formed protrusions. (H, I, J) Wild-type and CDCP1 -knockout MDCK cysts were incubated in the presence of HGF (50 ng/ml) for 1 d. (H) Ki67 was visualized with an Alexa Fluor 488-conjugated antibody (green), and actin filaments were stained with Alexa Fluor 594-phalloidin (magenta); arrowheads indicate transiently formed protrusions. (I) Diameter (μm) of cysts (n = 100). (J) Fraction of the total number of cysts counted (n > 100) with protrusions. Scale bars: 10 μm. Data information: In (B, C, D, I, J), the mean ratios ± SD were obtained from three independent experiments. * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001; NS, not significantly different; two-way ANOVA was calculated compared with HGF-treated cysts.

    Article Snippet: The following primary antibodies were used in this study: anti-CDCP1 (4115), anti-CDCP1 pY734 (9050), anti-STAT3 (9132), anti-STAT3 pY705 (9145), anti-myc-tag (2276), anti-Met pY1234/1235 (3077), anti-Met pY1349 (3121) anti-Met (3127, clone 25H2), and anti-Met (8198) antibodies were all purchased from Cell Signaling Technologies, anti-CDCP1 antibody (LC-C172540) was purchased from LSBio, anti-SFK (sc-18, clone SRC2), anti-ERα (sc-542, clone MC-20), anti-Gapdh (sc-32233, clone 6C5), and anti-MMP2 (sc-10736) antibodies were all purchased from Santa Cruz Biotechnology, anti-Src pY418 (44-660G), anti-Src pY529 (44-662G), and anti-Ki67 (14-5698-82) antibodies were all purchased from Thermo Fisher Scientific, anti-Src (OP07, clone Ab-1), anti-phosphotyrosine (05-1050, clone 4G10), and anti-MMP9 (444236) antibodies were all purchased from Millipore, anti-Fyn (610163, clone 25), anti-Lyn (610003, clone 42), and anti-Yes (610375, clone 1) were all purchased from BD Bioscience, anti-collagen IV antibody (ab6586) was purchased from Abcam, anti-laminin antibody (L9393) was purchased from Sigma-Aldrich, and anti-keratin 14 antibody (PRB-155P, clone AF64) was purchased from Covance.

    Techniques: Incubation, Staining, Knock-Out

    (A) Schematic structure of CDCP1. In the cytoplasmic region, CDCP1 contains a Src association motif around Tyr734 and two palmitoylation sites (Cys residues 689 and 690), which are required for the localization of CDCP1 in lipid rafts. Upon phosphorylation at Tyr734 by Src, CDCP1 is additionally phosphorylated at Tyr762, resulting in direct association with PKCδ, which promotes cell migration. In the extracellular region, CDCP1 contains three CUB domains that are required for protein–protein interactions. CDCP1 also harbors a proteolytic cleavage (shedding) site between the first and second CUB domains and can, thus, be present in either the full-length or cleaved form depending on the cellular context. TM, transmembrane domain. (B) Schematic diagram of CRISPR/Cas9-based generation of CDCP1 -knockout MDCK cells. Blue indicates the PAM sequence and bold letters indicate the start codon. The red arrowhead indicates the cleavage site. (C) Immunoblotting analysis of CDCP1 -knockout MDCK cells. Lysates from wild-type and CDCP1 -knockout MDCK cells were subjected to immunoblotting using the indicated antibodies. (D) CDCP1 -knockout MDCK cysts were incubated in the presence of hepatocyte growth factor (50 ng/ml) for 4 and 6 d. Ki67 was visualized with an Alexa Fluor 488–conjugated antibody (green) and actin filaments were stained with Alexa Fluor 594-phalloidin (magenta). Scale bars: 10 μm. Source data are available for this figure.

    Journal: Life Science Alliance

    Article Title: CDCP1 promotes compensatory renal growth by integrating Src and Met signaling

    doi: 10.26508/lsa.202000832

    Figure Lengend Snippet: (A) Schematic structure of CDCP1. In the cytoplasmic region, CDCP1 contains a Src association motif around Tyr734 and two palmitoylation sites (Cys residues 689 and 690), which are required for the localization of CDCP1 in lipid rafts. Upon phosphorylation at Tyr734 by Src, CDCP1 is additionally phosphorylated at Tyr762, resulting in direct association with PKCδ, which promotes cell migration. In the extracellular region, CDCP1 contains three CUB domains that are required for protein–protein interactions. CDCP1 also harbors a proteolytic cleavage (shedding) site between the first and second CUB domains and can, thus, be present in either the full-length or cleaved form depending on the cellular context. TM, transmembrane domain. (B) Schematic diagram of CRISPR/Cas9-based generation of CDCP1 -knockout MDCK cells. Blue indicates the PAM sequence and bold letters indicate the start codon. The red arrowhead indicates the cleavage site. (C) Immunoblotting analysis of CDCP1 -knockout MDCK cells. Lysates from wild-type and CDCP1 -knockout MDCK cells were subjected to immunoblotting using the indicated antibodies. (D) CDCP1 -knockout MDCK cysts were incubated in the presence of hepatocyte growth factor (50 ng/ml) for 4 and 6 d. Ki67 was visualized with an Alexa Fluor 488–conjugated antibody (green) and actin filaments were stained with Alexa Fluor 594-phalloidin (magenta). Scale bars: 10 μm. Source data are available for this figure.

    Article Snippet: The following primary antibodies were used in this study: anti-CDCP1 (4115), anti-CDCP1 pY734 (9050), anti-STAT3 (9132), anti-STAT3 pY705 (9145), anti-myc-tag (2276), anti-Met pY1234/1235 (3077), anti-Met pY1349 (3121) anti-Met (3127, clone 25H2), and anti-Met (8198) antibodies were all purchased from Cell Signaling Technologies, anti-CDCP1 antibody (LC-C172540) was purchased from LSBio, anti-SFK (sc-18, clone SRC2), anti-ERα (sc-542, clone MC-20), anti-Gapdh (sc-32233, clone 6C5), and anti-MMP2 (sc-10736) antibodies were all purchased from Santa Cruz Biotechnology, anti-Src pY418 (44-660G), anti-Src pY529 (44-662G), and anti-Ki67 (14-5698-82) antibodies were all purchased from Thermo Fisher Scientific, anti-Src (OP07, clone Ab-1), anti-phosphotyrosine (05-1050, clone 4G10), and anti-MMP9 (444236) antibodies were all purchased from Millipore, anti-Fyn (610163, clone 25), anti-Lyn (610003, clone 42), and anti-Yes (610375, clone 1) were all purchased from BD Bioscience, anti-collagen IV antibody (ab6586) was purchased from Abcam, anti-laminin antibody (L9393) was purchased from Sigma-Aldrich, and anti-keratin 14 antibody (PRB-155P, clone AF64) was purchased from Covance.

    Techniques: Migration, CRISPR, Knock-Out, Sequencing, Western Blot, Incubation, Staining

    (A) TRE-CDCP1-EGFP (CDCP1-EGFP cells)– and mutant (YF-EGFP or CG-EGFP)–harboring MDCK cells were incubated in the presence of Dox (1 μg/ml) for the indicated time periods. Cell lysates were subjected to immunoblotting using the indicated antibodies. (B) CDCP1-EGFP cells were incubated in the presence of Dox (1 μg/ml) for the indicated time periods. Actin filaments were stained with Alexa Fluor 594-phalloidin (magenta). Scale bars indicate 50 μm. (C) CDCP1-EGFP– and mutant-harboring MDCK cells were incubated in the presence of Dox (1 μg/ml) for 48 h. Detergent-resistant membrane (DRM) and non-DRM fractions were separated on a sucrose density gradient. Aliquots of the fractions were subjected to immunoblotting analysis using the indicated antibodies. (D) DRM fractions of CDCP1-myc–overexpressing MDCK cells were subjected to immunoprecipitation with an anti-myc-tag, anti-Src, anti-Lyn, or anti-Yes antibody. Immunoprecipitates were subjected to immunoblotting using the indicated antibodies. IgG HC, IgG heavy chain. (E) A list of SFK proteins found in DRM fractions. DRM fractions of MDCK cells were subjected to SDS–PAGE and silver staining. All proteins within polyacrylamide gel were trypsinized and subjected to mass spectrometry. Proteins were identified using the SwissProt database. (F, G) DRM and non-DRM fractions of CDCP1-myc-overexpressing MDCK cells were subjected to immunoblotting analysis using the indicated antibodies. (G) Relative amount of SFK was calculated by setting the mean value for mock-transfected cells to one. Data information: In (G), the mean ratios ± SD were obtained from three independent experiments. ** P < 0.01; *** P < 0.001; NS, not significantly different; unpaired two-tailed t test. Source data are available for this figure.

    Journal: Life Science Alliance

    Article Title: CDCP1 promotes compensatory renal growth by integrating Src and Met signaling

    doi: 10.26508/lsa.202000832

    Figure Lengend Snippet: (A) TRE-CDCP1-EGFP (CDCP1-EGFP cells)– and mutant (YF-EGFP or CG-EGFP)–harboring MDCK cells were incubated in the presence of Dox (1 μg/ml) for the indicated time periods. Cell lysates were subjected to immunoblotting using the indicated antibodies. (B) CDCP1-EGFP cells were incubated in the presence of Dox (1 μg/ml) for the indicated time periods. Actin filaments were stained with Alexa Fluor 594-phalloidin (magenta). Scale bars indicate 50 μm. (C) CDCP1-EGFP– and mutant-harboring MDCK cells were incubated in the presence of Dox (1 μg/ml) for 48 h. Detergent-resistant membrane (DRM) and non-DRM fractions were separated on a sucrose density gradient. Aliquots of the fractions were subjected to immunoblotting analysis using the indicated antibodies. (D) DRM fractions of CDCP1-myc–overexpressing MDCK cells were subjected to immunoprecipitation with an anti-myc-tag, anti-Src, anti-Lyn, or anti-Yes antibody. Immunoprecipitates were subjected to immunoblotting using the indicated antibodies. IgG HC, IgG heavy chain. (E) A list of SFK proteins found in DRM fractions. DRM fractions of MDCK cells were subjected to SDS–PAGE and silver staining. All proteins within polyacrylamide gel were trypsinized and subjected to mass spectrometry. Proteins were identified using the SwissProt database. (F, G) DRM and non-DRM fractions of CDCP1-myc-overexpressing MDCK cells were subjected to immunoblotting analysis using the indicated antibodies. (G) Relative amount of SFK was calculated by setting the mean value for mock-transfected cells to one. Data information: In (G), the mean ratios ± SD were obtained from three independent experiments. ** P < 0.01; *** P < 0.001; NS, not significantly different; unpaired two-tailed t test. Source data are available for this figure.

    Article Snippet: The following primary antibodies were used in this study: anti-CDCP1 (4115), anti-CDCP1 pY734 (9050), anti-STAT3 (9132), anti-STAT3 pY705 (9145), anti-myc-tag (2276), anti-Met pY1234/1235 (3077), anti-Met pY1349 (3121) anti-Met (3127, clone 25H2), and anti-Met (8198) antibodies were all purchased from Cell Signaling Technologies, anti-CDCP1 antibody (LC-C172540) was purchased from LSBio, anti-SFK (sc-18, clone SRC2), anti-ERα (sc-542, clone MC-20), anti-Gapdh (sc-32233, clone 6C5), and anti-MMP2 (sc-10736) antibodies were all purchased from Santa Cruz Biotechnology, anti-Src pY418 (44-660G), anti-Src pY529 (44-662G), and anti-Ki67 (14-5698-82) antibodies were all purchased from Thermo Fisher Scientific, anti-Src (OP07, clone Ab-1), anti-phosphotyrosine (05-1050, clone 4G10), and anti-MMP9 (444236) antibodies were all purchased from Millipore, anti-Fyn (610163, clone 25), anti-Lyn (610003, clone 42), and anti-Yes (610375, clone 1) were all purchased from BD Bioscience, anti-collagen IV antibody (ab6586) was purchased from Abcam, anti-laminin antibody (L9393) was purchased from Sigma-Aldrich, and anti-keratin 14 antibody (PRB-155P, clone AF64) was purchased from Covance.

    Techniques: Mutagenesis, Incubation, Western Blot, Staining, Immunoprecipitation, SDS Page, Silver Staining, Mass Spectrometry, Transfection, Two Tailed Test

    (A) Schematic illustration of analysis of TRE-CDCP1-EGFP-harboring MDCK cysts (CDCP1-EGFP cysts). TRE-CDCP1-EGFP-harboring cells were cultured within a collagen matrix for 5 d for cyst formation, and then CDCP1-EGFP cysts were incubated in the presence of Dox for ∼4 d. (B) CDCP1-EGFP cysts embedded within the collagen matrix were incubated in the presence of Dox (1 μg/ml) for the indicated time periods. Arrowheads indicate protruding cells and open arrowheads indicate multi-layered structures. (C) CDCP1-EGFP cysts were incubated in the presence of Dox (1 μg/ml) for 2 d. Activated Src was visualized with a Src pY418 antibody (magenta). (D, E) CDCP1-EGFP cysts embedded within the collagen matrix were pretreated with 20 nM dasatinib or 10 μM saracatinib for 2 h, and then incubated with Dox (1 μg/ml) for 4 d. (E) Fraction of the total number of cysts counted (n > 150) with protrusions. (F, G) CDCP1-YF-EGFP and CDCP1-CG-EGFP cysts embedded within the collagen matrix were incubated in the presence of Dox (1 μg/ml) for 4 d. (G) Fraction of the total number of cysts counted (n > 150) with protrusions. (H) CDCP1-EGFP cysts embedded within the collagen matrix were pretreated with 1 mM MβCD for 2 h, and then incubated with Dox (1 μg/ml) for 4 d. Actin filaments were stained with Alexa Fluor 594-phalloidin (magenta). Scale bars: 50 μm. Data information: In (E, G), the mean ratios ± SD were obtained from three independent experiments. *** P < 0.001; **** P < 0.0001; NS, not significantly different; two-way ANOVA compared with the Dox-treated CDCP1-EGFP cysts.

    Journal: Life Science Alliance

    Article Title: CDCP1 promotes compensatory renal growth by integrating Src and Met signaling

    doi: 10.26508/lsa.202000832

    Figure Lengend Snippet: (A) Schematic illustration of analysis of TRE-CDCP1-EGFP-harboring MDCK cysts (CDCP1-EGFP cysts). TRE-CDCP1-EGFP-harboring cells were cultured within a collagen matrix for 5 d for cyst formation, and then CDCP1-EGFP cysts were incubated in the presence of Dox for ∼4 d. (B) CDCP1-EGFP cysts embedded within the collagen matrix were incubated in the presence of Dox (1 μg/ml) for the indicated time periods. Arrowheads indicate protruding cells and open arrowheads indicate multi-layered structures. (C) CDCP1-EGFP cysts were incubated in the presence of Dox (1 μg/ml) for 2 d. Activated Src was visualized with a Src pY418 antibody (magenta). (D, E) CDCP1-EGFP cysts embedded within the collagen matrix were pretreated with 20 nM dasatinib or 10 μM saracatinib for 2 h, and then incubated with Dox (1 μg/ml) for 4 d. (E) Fraction of the total number of cysts counted (n > 150) with protrusions. (F, G) CDCP1-YF-EGFP and CDCP1-CG-EGFP cysts embedded within the collagen matrix were incubated in the presence of Dox (1 μg/ml) for 4 d. (G) Fraction of the total number of cysts counted (n > 150) with protrusions. (H) CDCP1-EGFP cysts embedded within the collagen matrix were pretreated with 1 mM MβCD for 2 h, and then incubated with Dox (1 μg/ml) for 4 d. Actin filaments were stained with Alexa Fluor 594-phalloidin (magenta). Scale bars: 50 μm. Data information: In (E, G), the mean ratios ± SD were obtained from three independent experiments. *** P < 0.001; **** P < 0.0001; NS, not significantly different; two-way ANOVA compared with the Dox-treated CDCP1-EGFP cysts.

    Article Snippet: The following primary antibodies were used in this study: anti-CDCP1 (4115), anti-CDCP1 pY734 (9050), anti-STAT3 (9132), anti-STAT3 pY705 (9145), anti-myc-tag (2276), anti-Met pY1234/1235 (3077), anti-Met pY1349 (3121) anti-Met (3127, clone 25H2), and anti-Met (8198) antibodies were all purchased from Cell Signaling Technologies, anti-CDCP1 antibody (LC-C172540) was purchased from LSBio, anti-SFK (sc-18, clone SRC2), anti-ERα (sc-542, clone MC-20), anti-Gapdh (sc-32233, clone 6C5), and anti-MMP2 (sc-10736) antibodies were all purchased from Santa Cruz Biotechnology, anti-Src pY418 (44-660G), anti-Src pY529 (44-662G), and anti-Ki67 (14-5698-82) antibodies were all purchased from Thermo Fisher Scientific, anti-Src (OP07, clone Ab-1), anti-phosphotyrosine (05-1050, clone 4G10), and anti-MMP9 (444236) antibodies were all purchased from Millipore, anti-Fyn (610163, clone 25), anti-Lyn (610003, clone 42), and anti-Yes (610375, clone 1) were all purchased from BD Bioscience, anti-collagen IV antibody (ab6586) was purchased from Abcam, anti-laminin antibody (L9393) was purchased from Sigma-Aldrich, and anti-keratin 14 antibody (PRB-155P, clone AF64) was purchased from Covance.

    Techniques: Cell Culture, Incubation, Staining

    (A) Gene Ontology analysis of CDCP1-EGFP cysts. Purple bars indicate signaling related to Gene Ontology annotations. Magenta and blue bars indicate morphogenesis and cell proliferation, respectively. (B) STAT3 upstream regulatory networks of CDCP1-EGFP and CDCP1-CG-EGFP cysts. Activation Z-scores are presented in Tables S2 and S3. Red and green symbols indicate transcript levels up-regulated and down-regulated by CDCP1 overexpression, respectively. Arrows indicate activation (orange), inhibition (blue), inconsistency with the state of the downstream molecule (yellow) and unknown effect (gray). (C) Heat map representing changes in the expression of STAT3 target genes in CDCP1-EGFP (WT) and CDCP1-CG-EGFP (CG) cysts.

    Journal: Life Science Alliance

    Article Title: CDCP1 promotes compensatory renal growth by integrating Src and Met signaling

    doi: 10.26508/lsa.202000832

    Figure Lengend Snippet: (A) Gene Ontology analysis of CDCP1-EGFP cysts. Purple bars indicate signaling related to Gene Ontology annotations. Magenta and blue bars indicate morphogenesis and cell proliferation, respectively. (B) STAT3 upstream regulatory networks of CDCP1-EGFP and CDCP1-CG-EGFP cysts. Activation Z-scores are presented in Tables S2 and S3. Red and green symbols indicate transcript levels up-regulated and down-regulated by CDCP1 overexpression, respectively. Arrows indicate activation (orange), inhibition (blue), inconsistency with the state of the downstream molecule (yellow) and unknown effect (gray). (C) Heat map representing changes in the expression of STAT3 target genes in CDCP1-EGFP (WT) and CDCP1-CG-EGFP (CG) cysts.

    Article Snippet: The following primary antibodies were used in this study: anti-CDCP1 (4115), anti-CDCP1 pY734 (9050), anti-STAT3 (9132), anti-STAT3 pY705 (9145), anti-myc-tag (2276), anti-Met pY1234/1235 (3077), anti-Met pY1349 (3121) anti-Met (3127, clone 25H2), and anti-Met (8198) antibodies were all purchased from Cell Signaling Technologies, anti-CDCP1 antibody (LC-C172540) was purchased from LSBio, anti-SFK (sc-18, clone SRC2), anti-ERα (sc-542, clone MC-20), anti-Gapdh (sc-32233, clone 6C5), and anti-MMP2 (sc-10736) antibodies were all purchased from Santa Cruz Biotechnology, anti-Src pY418 (44-660G), anti-Src pY529 (44-662G), and anti-Ki67 (14-5698-82) antibodies were all purchased from Thermo Fisher Scientific, anti-Src (OP07, clone Ab-1), anti-phosphotyrosine (05-1050, clone 4G10), and anti-MMP9 (444236) antibodies were all purchased from Millipore, anti-Fyn (610163, clone 25), anti-Lyn (610003, clone 42), and anti-Yes (610375, clone 1) were all purchased from BD Bioscience, anti-collagen IV antibody (ab6586) was purchased from Abcam, anti-laminin antibody (L9393) was purchased from Sigma-Aldrich, and anti-keratin 14 antibody (PRB-155P, clone AF64) was purchased from Covance.

    Techniques: Activation Assay, Over Expression, Inhibition, Expressing

    (A) CDCP1-myc– and mutant-overexpressing MDCK cells were embedded within the collagen matrix and cultured for 9 d. Cyst lysates were subjected to immunoblotting using the indicated antibodies. (B) CDCP1-EGFP cysts were incubated with Dox (1 μg/ml) for 2 or 4 d, and then subjected to quantitative real-time PCR. Relative mRNA expression levels were calculated by setting the mean value for non-treated cysts to one. (C) CDCP1-EGFP cysts were pretreated with the indicated STAT3-specific inhibitors for 2 h and then incubated with Dox (1 μg/ml) for 4 d. (D, E) STAT3-Y705F–overexpressing CDCP1-EGFP cysts were incubated with Dox (1 μg/ml) for 4 d. (D) Actin filaments were stained with Alexa Fluor 594-phalloidin (magenta). Scale bars: 50 μm. (E) Fraction of the total number of cysts counted (n > 150) with protrusions. (F, G, H) MDCK cysts embedded within the collagen matrix were pretreated with S3i-201 (+ S3i, 50 nM; ++ S3i, 100 nM) or stattic (+ Sta, 2.5 nM; ++ Sta, 5.0 nM) for 2 h and incubated in the presence of hepatocyte growth factor (50 ng/ml) for 1 d. (F) Diameter (μm) of cysts (n = 100). (G) Ratio of Ki67-positive cell in cyst (n = 50). (H) Fraction of the total number of cysts counted (n > 100) with protrusions. Dotted blue indicates the average diameter of non-treated cysts. Data information: In (B, E, F, G, H), the mean ratios ± SD were obtained from three independent experiments. * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001; NS, not significantly different; ANOVA was calculated compared with the non-treated cyst (B) or the hepatocyte growth factor-treated cysts (F, G, H); unpaired two-tailed t test (E). Source data are available for this figure.

    Journal: Life Science Alliance

    Article Title: CDCP1 promotes compensatory renal growth by integrating Src and Met signaling

    doi: 10.26508/lsa.202000832

    Figure Lengend Snippet: (A) CDCP1-myc– and mutant-overexpressing MDCK cells were embedded within the collagen matrix and cultured for 9 d. Cyst lysates were subjected to immunoblotting using the indicated antibodies. (B) CDCP1-EGFP cysts were incubated with Dox (1 μg/ml) for 2 or 4 d, and then subjected to quantitative real-time PCR. Relative mRNA expression levels were calculated by setting the mean value for non-treated cysts to one. (C) CDCP1-EGFP cysts were pretreated with the indicated STAT3-specific inhibitors for 2 h and then incubated with Dox (1 μg/ml) for 4 d. (D, E) STAT3-Y705F–overexpressing CDCP1-EGFP cysts were incubated with Dox (1 μg/ml) for 4 d. (D) Actin filaments were stained with Alexa Fluor 594-phalloidin (magenta). Scale bars: 50 μm. (E) Fraction of the total number of cysts counted (n > 150) with protrusions. (F, G, H) MDCK cysts embedded within the collagen matrix were pretreated with S3i-201 (+ S3i, 50 nM; ++ S3i, 100 nM) or stattic (+ Sta, 2.5 nM; ++ Sta, 5.0 nM) for 2 h and incubated in the presence of hepatocyte growth factor (50 ng/ml) for 1 d. (F) Diameter (μm) of cysts (n = 100). (G) Ratio of Ki67-positive cell in cyst (n = 50). (H) Fraction of the total number of cysts counted (n > 100) with protrusions. Dotted blue indicates the average diameter of non-treated cysts. Data information: In (B, E, F, G, H), the mean ratios ± SD were obtained from three independent experiments. * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001; NS, not significantly different; ANOVA was calculated compared with the non-treated cyst (B) or the hepatocyte growth factor-treated cysts (F, G, H); unpaired two-tailed t test (E). Source data are available for this figure.

    Article Snippet: The following primary antibodies were used in this study: anti-CDCP1 (4115), anti-CDCP1 pY734 (9050), anti-STAT3 (9132), anti-STAT3 pY705 (9145), anti-myc-tag (2276), anti-Met pY1234/1235 (3077), anti-Met pY1349 (3121) anti-Met (3127, clone 25H2), and anti-Met (8198) antibodies were all purchased from Cell Signaling Technologies, anti-CDCP1 antibody (LC-C172540) was purchased from LSBio, anti-SFK (sc-18, clone SRC2), anti-ERα (sc-542, clone MC-20), anti-Gapdh (sc-32233, clone 6C5), and anti-MMP2 (sc-10736) antibodies were all purchased from Santa Cruz Biotechnology, anti-Src pY418 (44-660G), anti-Src pY529 (44-662G), and anti-Ki67 (14-5698-82) antibodies were all purchased from Thermo Fisher Scientific, anti-Src (OP07, clone Ab-1), anti-phosphotyrosine (05-1050, clone 4G10), and anti-MMP9 (444236) antibodies were all purchased from Millipore, anti-Fyn (610163, clone 25), anti-Lyn (610003, clone 42), and anti-Yes (610375, clone 1) were all purchased from BD Bioscience, anti-collagen IV antibody (ab6586) was purchased from Abcam, anti-laminin antibody (L9393) was purchased from Sigma-Aldrich, and anti-keratin 14 antibody (PRB-155P, clone AF64) was purchased from Covance.

    Techniques: Mutagenesis, Cell Culture, Western Blot, Incubation, Real-time Polymerase Chain Reaction, Expressing, Staining, Two Tailed Test

    (A) CDCP1-EGFP cysts embedded within the collagen matrix were incubated with Dox (1 μg/ml) for the indicated time periods. The basement membrane was visualized with an anti-laminin antibody (magenta). The arrowheads indicate protruding cells, and the open arrowheads indicate multi-layered structures. (B, C) CDCP1-EGFP cysts were pretreated with 20 μM marimastat for 2 h, and then incubated with Dox (1 μg/ml) for 4 d. (B) The basement membrane was visualized using an anti-laminin antibody (magenta). (C) The histogram depicting the percentage of cells with protrusions was calculated by setting the total number of cysts to 100% (n > 150). (D, E, F) MDCK cysts embedded within the collagen matrix were pretreated with marimastat (+ Mari, 10 μM; ++ Mari, 20 μM) for 2 h, and then incubated in the presence of hepatocyte growth factor (50 ng/ml) for 1 d. (D) Diameter of cysts (μm) of cysts (n = 100). (E) Ratio of Ki67-positive cell in cyst (n = 50). (F) Fraction of the total number of cysts counted (n > 100) with protrusions. Dotted blue line indicates the average diameter of non-treated cysts. (G) TRE-CDCP1-mCherry–harboring MDCK cells were embedded within a 2% DQ-collagen-containing matrix. CDCP1-mCherry cysts were pretreated with 20 μM marimastat for 2 h, and then incubated with Dox (1 μg/ml) for 4 d. DQ fluorescence (green) was visualized under a fluorescent microscope. (H) A schematic diagram of STAT3-MER protein. A modified ligand-binding domain of estrogen receptor (MER) is fused to C-terminal region of full-length STAT3 protein. SH, Src homology domain. (I, J) STAT3-MER–overexpressing cysts embedded within the collagen matrix were incubated with 1 μM 4-OHT for 4 d. (I) STAT3-MER was visualized with ERα antibody (green) and actin filaments were stained with Alexa Fluor 594-phalloidin (magenta). (J) Fraction of the total number of cysts counted (n > 100) with protrusions. Scale bars: 50 μm. Data information: In (C, D, E, F, J), the mean ratios ± SD were obtained from three independent experiments. * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001; two-way ANOVA was calculated compared with the Dox-treated cysts (C) or the hepatocyte growth factor-treated cysts (D, E, F); unpaired two-tailed t test (J).

    Journal: Life Science Alliance

    Article Title: CDCP1 promotes compensatory renal growth by integrating Src and Met signaling

    doi: 10.26508/lsa.202000832

    Figure Lengend Snippet: (A) CDCP1-EGFP cysts embedded within the collagen matrix were incubated with Dox (1 μg/ml) for the indicated time periods. The basement membrane was visualized with an anti-laminin antibody (magenta). The arrowheads indicate protruding cells, and the open arrowheads indicate multi-layered structures. (B, C) CDCP1-EGFP cysts were pretreated with 20 μM marimastat for 2 h, and then incubated with Dox (1 μg/ml) for 4 d. (B) The basement membrane was visualized using an anti-laminin antibody (magenta). (C) The histogram depicting the percentage of cells with protrusions was calculated by setting the total number of cysts to 100% (n > 150). (D, E, F) MDCK cysts embedded within the collagen matrix were pretreated with marimastat (+ Mari, 10 μM; ++ Mari, 20 μM) for 2 h, and then incubated in the presence of hepatocyte growth factor (50 ng/ml) for 1 d. (D) Diameter of cysts (μm) of cysts (n = 100). (E) Ratio of Ki67-positive cell in cyst (n = 50). (F) Fraction of the total number of cysts counted (n > 100) with protrusions. Dotted blue line indicates the average diameter of non-treated cysts. (G) TRE-CDCP1-mCherry–harboring MDCK cells were embedded within a 2% DQ-collagen-containing matrix. CDCP1-mCherry cysts were pretreated with 20 μM marimastat for 2 h, and then incubated with Dox (1 μg/ml) for 4 d. DQ fluorescence (green) was visualized under a fluorescent microscope. (H) A schematic diagram of STAT3-MER protein. A modified ligand-binding domain of estrogen receptor (MER) is fused to C-terminal region of full-length STAT3 protein. SH, Src homology domain. (I, J) STAT3-MER–overexpressing cysts embedded within the collagen matrix were incubated with 1 μM 4-OHT for 4 d. (I) STAT3-MER was visualized with ERα antibody (green) and actin filaments were stained with Alexa Fluor 594-phalloidin (magenta). (J) Fraction of the total number of cysts counted (n > 100) with protrusions. Scale bars: 50 μm. Data information: In (C, D, E, F, J), the mean ratios ± SD were obtained from three independent experiments. * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001; two-way ANOVA was calculated compared with the Dox-treated cysts (C) or the hepatocyte growth factor-treated cysts (D, E, F); unpaired two-tailed t test (J).

    Article Snippet: The following primary antibodies were used in this study: anti-CDCP1 (4115), anti-CDCP1 pY734 (9050), anti-STAT3 (9132), anti-STAT3 pY705 (9145), anti-myc-tag (2276), anti-Met pY1234/1235 (3077), anti-Met pY1349 (3121) anti-Met (3127, clone 25H2), and anti-Met (8198) antibodies were all purchased from Cell Signaling Technologies, anti-CDCP1 antibody (LC-C172540) was purchased from LSBio, anti-SFK (sc-18, clone SRC2), anti-ERα (sc-542, clone MC-20), anti-Gapdh (sc-32233, clone 6C5), and anti-MMP2 (sc-10736) antibodies were all purchased from Santa Cruz Biotechnology, anti-Src pY418 (44-660G), anti-Src pY529 (44-662G), and anti-Ki67 (14-5698-82) antibodies were all purchased from Thermo Fisher Scientific, anti-Src (OP07, clone Ab-1), anti-phosphotyrosine (05-1050, clone 4G10), and anti-MMP9 (444236) antibodies were all purchased from Millipore, anti-Fyn (610163, clone 25), anti-Lyn (610003, clone 42), and anti-Yes (610375, clone 1) were all purchased from BD Bioscience, anti-collagen IV antibody (ab6586) was purchased from Abcam, anti-laminin antibody (L9393) was purchased from Sigma-Aldrich, and anti-keratin 14 antibody (PRB-155P, clone AF64) was purchased from Covance.

    Techniques: Incubation, Fluorescence, Microscopy, Modification, Ligand Binding Assay, Staining, Two Tailed Test

    (A) Wild-type and CDCP1 -knockout TRE-Met harboring MDCK cells embedded within the collagen matrix were incubated with hepatocyte growth factor (50 ng/ml) or Dox (1 μg/ml) for 4 d. Met was visualized with a Met antibody (green) and actin filaments were stained with Alexa Fluor 594-phalloidin (magenta). Scale bars indicate 50 μm. (B) Renal cancer cell lines, A498 and ACHN, were incubated in the presence or absence of hepatocyte growth factor (50 ng/ml) for 30 min. Cell lysates were subjected to immunoprecipitation with anti-CDCP1 or anti-Met antibody. Immunoprecipitates were subjected to immunoblotting using the indicated antibodies. IgG HC, IgG heavy chain. Source data are available for this figure.

    Journal: Life Science Alliance

    Article Title: CDCP1 promotes compensatory renal growth by integrating Src and Met signaling

    doi: 10.26508/lsa.202000832

    Figure Lengend Snippet: (A) Wild-type and CDCP1 -knockout TRE-Met harboring MDCK cells embedded within the collagen matrix were incubated with hepatocyte growth factor (50 ng/ml) or Dox (1 μg/ml) for 4 d. Met was visualized with a Met antibody (green) and actin filaments were stained with Alexa Fluor 594-phalloidin (magenta). Scale bars indicate 50 μm. (B) Renal cancer cell lines, A498 and ACHN, were incubated in the presence or absence of hepatocyte growth factor (50 ng/ml) for 30 min. Cell lysates were subjected to immunoprecipitation with anti-CDCP1 or anti-Met antibody. Immunoprecipitates were subjected to immunoblotting using the indicated antibodies. IgG HC, IgG heavy chain. Source data are available for this figure.

    Article Snippet: The following primary antibodies were used in this study: anti-CDCP1 (4115), anti-CDCP1 pY734 (9050), anti-STAT3 (9132), anti-STAT3 pY705 (9145), anti-myc-tag (2276), anti-Met pY1234/1235 (3077), anti-Met pY1349 (3121) anti-Met (3127, clone 25H2), and anti-Met (8198) antibodies were all purchased from Cell Signaling Technologies, anti-CDCP1 antibody (LC-C172540) was purchased from LSBio, anti-SFK (sc-18, clone SRC2), anti-ERα (sc-542, clone MC-20), anti-Gapdh (sc-32233, clone 6C5), and anti-MMP2 (sc-10736) antibodies were all purchased from Santa Cruz Biotechnology, anti-Src pY418 (44-660G), anti-Src pY529 (44-662G), and anti-Ki67 (14-5698-82) antibodies were all purchased from Thermo Fisher Scientific, anti-Src (OP07, clone Ab-1), anti-phosphotyrosine (05-1050, clone 4G10), and anti-MMP9 (444236) antibodies were all purchased from Millipore, anti-Fyn (610163, clone 25), anti-Lyn (610003, clone 42), and anti-Yes (610375, clone 1) were all purchased from BD Bioscience, anti-collagen IV antibody (ab6586) was purchased from Abcam, anti-laminin antibody (L9393) was purchased from Sigma-Aldrich, and anti-keratin 14 antibody (PRB-155P, clone AF64) was purchased from Covance.

    Techniques: Knock-Out, Incubation, Staining, Immunoprecipitation, Western Blot

    (A) CDCP1-EGFP cysts were pretreated with the indicated Met-specific inhibitors for 2 h and then incubated with Dox (1 μg/ml) for 4 d. (B, C) Schematic representation of CDCP1 and the deletion mutants. (B) SP, signal peptide; CUB, CUB domain; TM, transmembrane domain; CD, cytosolic domain. Lysates from HEK293 cells overexpressing both CDCP1-myc and Met were subjected to immunoprecipitation with an anti-myc-tag antibody. Immunoprecipitates were subjected to immunoblotting using the indicated antibodies. IgG HC, IgG heavy chain. (D) CDCP1-myc- and CDCP1-PR-myc-overexpressing MDCK cells were embedded within the collagen matrix and cultured for 9 d. Cyst lysates were subjected to immunoblotting analysis using the indicated antibodies. (E, F) CDCP1-PR-EGFP cysts embedded within the collagen matrix were incubated with Dox (1 μg/ml) for 4 d. (E) Actin filaments were stained with Alexa Fluor 594-phalloidin (magenta). Scale bars: 50 μm. (F) Fraction of the total number of cysts counted (n > 150) with protrusions. Data information: In (F), the mean ratios ± SD were obtained from three independent experiments. ** P < 0.01; **** P < 0.0001; NS, not significantly different; two-way ANOVA was calculated compared with the Dox-treated cysts. Source data are available for this figure.

    Journal: Life Science Alliance

    Article Title: CDCP1 promotes compensatory renal growth by integrating Src and Met signaling

    doi: 10.26508/lsa.202000832

    Figure Lengend Snippet: (A) CDCP1-EGFP cysts were pretreated with the indicated Met-specific inhibitors for 2 h and then incubated with Dox (1 μg/ml) for 4 d. (B, C) Schematic representation of CDCP1 and the deletion mutants. (B) SP, signal peptide; CUB, CUB domain; TM, transmembrane domain; CD, cytosolic domain. Lysates from HEK293 cells overexpressing both CDCP1-myc and Met were subjected to immunoprecipitation with an anti-myc-tag antibody. Immunoprecipitates were subjected to immunoblotting using the indicated antibodies. IgG HC, IgG heavy chain. (D) CDCP1-myc- and CDCP1-PR-myc-overexpressing MDCK cells were embedded within the collagen matrix and cultured for 9 d. Cyst lysates were subjected to immunoblotting analysis using the indicated antibodies. (E, F) CDCP1-PR-EGFP cysts embedded within the collagen matrix were incubated with Dox (1 μg/ml) for 4 d. (E) Actin filaments were stained with Alexa Fluor 594-phalloidin (magenta). Scale bars: 50 μm. (F) Fraction of the total number of cysts counted (n > 150) with protrusions. Data information: In (F), the mean ratios ± SD were obtained from three independent experiments. ** P < 0.01; **** P < 0.0001; NS, not significantly different; two-way ANOVA was calculated compared with the Dox-treated cysts. Source data are available for this figure.

    Article Snippet: The following primary antibodies were used in this study: anti-CDCP1 (4115), anti-CDCP1 pY734 (9050), anti-STAT3 (9132), anti-STAT3 pY705 (9145), anti-myc-tag (2276), anti-Met pY1234/1235 (3077), anti-Met pY1349 (3121) anti-Met (3127, clone 25H2), and anti-Met (8198) antibodies were all purchased from Cell Signaling Technologies, anti-CDCP1 antibody (LC-C172540) was purchased from LSBio, anti-SFK (sc-18, clone SRC2), anti-ERα (sc-542, clone MC-20), anti-Gapdh (sc-32233, clone 6C5), and anti-MMP2 (sc-10736) antibodies were all purchased from Santa Cruz Biotechnology, anti-Src pY418 (44-660G), anti-Src pY529 (44-662G), and anti-Ki67 (14-5698-82) antibodies were all purchased from Thermo Fisher Scientific, anti-Src (OP07, clone Ab-1), anti-phosphotyrosine (05-1050, clone 4G10), and anti-MMP9 (444236) antibodies were all purchased from Millipore, anti-Fyn (610163, clone 25), anti-Lyn (610003, clone 42), and anti-Yes (610375, clone 1) were all purchased from BD Bioscience, anti-collagen IV antibody (ab6586) was purchased from Abcam, anti-laminin antibody (L9393) was purchased from Sigma-Aldrich, and anti-keratin 14 antibody (PRB-155P, clone AF64) was purchased from Covance.

    Techniques: Incubation, Immunoprecipitation, Western Blot, Cell Culture, Staining

    (A) CDCP1-EGFP, CDCP1-YF-EGFP, and CDCP1-PR-EGFP cells were incubated with Dox (1 μg/ml) for 2, 4 d. Cell lysates were subjected to immunoblotting analysis using the indicated analysis using the indicated antibodies. (B, C) CDCP1-EGFP cells were incubated with Dox (1 μg/ml) for 2, 4 d. NK4 (1 μg/ml) were added 2 h before the Dox treatment. Saracatinib (10 μM, Sara) or Met/Ron kinase inhibitor (100 nM, Met inh.) were added 3 d after the Dox treatment. Met were stained with Alexa Fluor 594-phalloidin (magenta). Scale bars: 20 μm. (C) Ratio of plasma membrane-localized Met was calculated (n = 50). (D) CDCP1-EGFP cells were incubated with Dox (1 μg/ml) for 4 d. NK4 (1 μg/ml) were added 2 h before the Dox treatment. Saracatinib (+ Sara, 10 μM; ++ Sara 20 μM) were added 3 d after the Dox treatment. Cell lysates were subjected to immunoblotting analysis using the indicated analysis using the indicated antibodies. (E, F) CDCP1-EGFP cells were incubated with Dox (1 μg/ml) for 4 d. Saracatinib (10 μM, Sara) were added 3 d after the Dox treatment. Met/Ron kinase inhibitor (100 nM, Met inh.) were added 30 min before cell extraction. Cell lysates were subjected to immunoprecipitation with an anti-Met antibody, and immunoprecipitates were subjected to immunoblotting using the indicated antibodies. IgG HC, IgG heavy chain. (F) Relative ratio of associated STAT3 was calculated by setting the mean value for non-treated cells to one. (G) Schematic diagram of the CDCP1-Src-Met complex-mediated STAT3 activation. Data information: In (C, F), the mean ratios ± SD were obtained from three independent experiments. * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001; NS, not significantly different; two-way ANOVA was calculated compared with the Dox-treated cells (C); unpaired two-tailed t test (F). Source data are available for this figure.

    Journal: Life Science Alliance

    Article Title: CDCP1 promotes compensatory renal growth by integrating Src and Met signaling

    doi: 10.26508/lsa.202000832

    Figure Lengend Snippet: (A) CDCP1-EGFP, CDCP1-YF-EGFP, and CDCP1-PR-EGFP cells were incubated with Dox (1 μg/ml) for 2, 4 d. Cell lysates were subjected to immunoblotting analysis using the indicated analysis using the indicated antibodies. (B, C) CDCP1-EGFP cells were incubated with Dox (1 μg/ml) for 2, 4 d. NK4 (1 μg/ml) were added 2 h before the Dox treatment. Saracatinib (10 μM, Sara) or Met/Ron kinase inhibitor (100 nM, Met inh.) were added 3 d after the Dox treatment. Met were stained with Alexa Fluor 594-phalloidin (magenta). Scale bars: 20 μm. (C) Ratio of plasma membrane-localized Met was calculated (n = 50). (D) CDCP1-EGFP cells were incubated with Dox (1 μg/ml) for 4 d. NK4 (1 μg/ml) were added 2 h before the Dox treatment. Saracatinib (+ Sara, 10 μM; ++ Sara 20 μM) were added 3 d after the Dox treatment. Cell lysates were subjected to immunoblotting analysis using the indicated analysis using the indicated antibodies. (E, F) CDCP1-EGFP cells were incubated with Dox (1 μg/ml) for 4 d. Saracatinib (10 μM, Sara) were added 3 d after the Dox treatment. Met/Ron kinase inhibitor (100 nM, Met inh.) were added 30 min before cell extraction. Cell lysates were subjected to immunoprecipitation with an anti-Met antibody, and immunoprecipitates were subjected to immunoblotting using the indicated antibodies. IgG HC, IgG heavy chain. (F) Relative ratio of associated STAT3 was calculated by setting the mean value for non-treated cells to one. (G) Schematic diagram of the CDCP1-Src-Met complex-mediated STAT3 activation. Data information: In (C, F), the mean ratios ± SD were obtained from three independent experiments. * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001; NS, not significantly different; two-way ANOVA was calculated compared with the Dox-treated cells (C); unpaired two-tailed t test (F). Source data are available for this figure.

    Article Snippet: The following primary antibodies were used in this study: anti-CDCP1 (4115), anti-CDCP1 pY734 (9050), anti-STAT3 (9132), anti-STAT3 pY705 (9145), anti-myc-tag (2276), anti-Met pY1234/1235 (3077), anti-Met pY1349 (3121) anti-Met (3127, clone 25H2), and anti-Met (8198) antibodies were all purchased from Cell Signaling Technologies, anti-CDCP1 antibody (LC-C172540) was purchased from LSBio, anti-SFK (sc-18, clone SRC2), anti-ERα (sc-542, clone MC-20), anti-Gapdh (sc-32233, clone 6C5), and anti-MMP2 (sc-10736) antibodies were all purchased from Santa Cruz Biotechnology, anti-Src pY418 (44-660G), anti-Src pY529 (44-662G), and anti-Ki67 (14-5698-82) antibodies were all purchased from Thermo Fisher Scientific, anti-Src (OP07, clone Ab-1), anti-phosphotyrosine (05-1050, clone 4G10), and anti-MMP9 (444236) antibodies were all purchased from Millipore, anti-Fyn (610163, clone 25), anti-Lyn (610003, clone 42), and anti-Yes (610375, clone 1) were all purchased from BD Bioscience, anti-collagen IV antibody (ab6586) was purchased from Abcam, anti-laminin antibody (L9393) was purchased from Sigma-Aldrich, and anti-keratin 14 antibody (PRB-155P, clone AF64) was purchased from Covance.

    Techniques: Incubation, Western Blot, Staining, Immunoprecipitation, Activation Assay, Two Tailed Test

    (A, B, C) Related to . Relative ratio of expression (A) and activation level of Met (B, C) was calculated by setting the mean value for non-treated cells to one. (D) CDCP1-EGFP and CDCP1-YF-EGFP cells were incubated with Dox (1 μg/ml) for 4 d. Detergent-resistant membrane and non-detergent-resistant membrane fractions were separated in a sucrose density gradient. Aliquots of the fractions were subjected to immunoblotting analysis using the indicated antibodies. (E, F, G) Related to . Relative ratio of expression (E) and activation level of Met (F, G) was calculated by setting the mean value for Dox-treated cells to one. (H) A schematic model of the role of CDCP1-Src in hepatocyte growth factor-induced morphogenesis. CDCP1-Src activates the Met-STAT3 signaling on lipid rafts, leading to outgrowth through induction of ECM rearrangement and cell growth/proliferation. The mammalian target of rapamycin pathway contributes to cell growth. (I, J, K) CDCP1-EGFP cells were incubated in medium supplemented with the presence of indicated FBS concentration with Dox (1 μg/ml) for 4 d. (I) Cell lysates were subjected to immunoblotting using the indicated antibodies. (J, K) Relative ratio of Met phosphorylation level was calculated by setting the mean value for non-treated cells to one. Data information: In (A, B, C, E, F, G, J, K), the mean ratios ± SD were obtained from at least three independent experiments. NS, not significantly different; two-way ANOVA was calculated compared with the non–Dox-treated cells (A, B, C) or the Dox-treated cells (E, F, G); unpaired two-tailed t test (J, K). Source data are available for this figure.

    Journal: Life Science Alliance

    Article Title: CDCP1 promotes compensatory renal growth by integrating Src and Met signaling

    doi: 10.26508/lsa.202000832

    Figure Lengend Snippet: (A, B, C) Related to . Relative ratio of expression (A) and activation level of Met (B, C) was calculated by setting the mean value for non-treated cells to one. (D) CDCP1-EGFP and CDCP1-YF-EGFP cells were incubated with Dox (1 μg/ml) for 4 d. Detergent-resistant membrane and non-detergent-resistant membrane fractions were separated in a sucrose density gradient. Aliquots of the fractions were subjected to immunoblotting analysis using the indicated antibodies. (E, F, G) Related to . Relative ratio of expression (E) and activation level of Met (F, G) was calculated by setting the mean value for Dox-treated cells to one. (H) A schematic model of the role of CDCP1-Src in hepatocyte growth factor-induced morphogenesis. CDCP1-Src activates the Met-STAT3 signaling on lipid rafts, leading to outgrowth through induction of ECM rearrangement and cell growth/proliferation. The mammalian target of rapamycin pathway contributes to cell growth. (I, J, K) CDCP1-EGFP cells were incubated in medium supplemented with the presence of indicated FBS concentration with Dox (1 μg/ml) for 4 d. (I) Cell lysates were subjected to immunoblotting using the indicated antibodies. (J, K) Relative ratio of Met phosphorylation level was calculated by setting the mean value for non-treated cells to one. Data information: In (A, B, C, E, F, G, J, K), the mean ratios ± SD were obtained from at least three independent experiments. NS, not significantly different; two-way ANOVA was calculated compared with the non–Dox-treated cells (A, B, C) or the Dox-treated cells (E, F, G); unpaired two-tailed t test (J, K). Source data are available for this figure.

    Article Snippet: The following primary antibodies were used in this study: anti-CDCP1 (4115), anti-CDCP1 pY734 (9050), anti-STAT3 (9132), anti-STAT3 pY705 (9145), anti-myc-tag (2276), anti-Met pY1234/1235 (3077), anti-Met pY1349 (3121) anti-Met (3127, clone 25H2), and anti-Met (8198) antibodies were all purchased from Cell Signaling Technologies, anti-CDCP1 antibody (LC-C172540) was purchased from LSBio, anti-SFK (sc-18, clone SRC2), anti-ERα (sc-542, clone MC-20), anti-Gapdh (sc-32233, clone 6C5), and anti-MMP2 (sc-10736) antibodies were all purchased from Santa Cruz Biotechnology, anti-Src pY418 (44-660G), anti-Src pY529 (44-662G), and anti-Ki67 (14-5698-82) antibodies were all purchased from Thermo Fisher Scientific, anti-Src (OP07, clone Ab-1), anti-phosphotyrosine (05-1050, clone 4G10), and anti-MMP9 (444236) antibodies were all purchased from Millipore, anti-Fyn (610163, clone 25), anti-Lyn (610003, clone 42), and anti-Yes (610375, clone 1) were all purchased from BD Bioscience, anti-collagen IV antibody (ab6586) was purchased from Abcam, anti-laminin antibody (L9393) was purchased from Sigma-Aldrich, and anti-keratin 14 antibody (PRB-155P, clone AF64) was purchased from Covance.

    Techniques: Expressing, Activation Assay, Incubation, Western Blot, Concentration Assay, Two Tailed Test

    (A) Schematic diagram of CRISPR/Cas9-based generation of Cdcp1 -knockout mouse. Blue indicates the PAM sequence and bold letters indicate the start codon. The red arrowhead indicates the cleavage site. (B) Representative picture of wild-type (+/+), Cdcp1 heterozygous (+/−) and homozygous knockout (−/−) littermate mice at 8 wk of age. (C, D) Validation of gene knockout was performed by genotyping (C) and Western blot (D). PCR verification of deletion at first exon of Cdcp1 gene using Fw and Rev primers depicted in panel (A). Lysates from mouse kidney were subjected to immunoblotting using the indicated antibodies. (E, F) Whole body (E) and kidney (F) weight of wild-type (+/+) and Cdcp1 homozygous knockout (−/−) mice at 8 wk of age. Data information: In (E, F), the mean ratios ± SD were obtained from 10 mice per group. NS, not significantly different; unpaired two-tailed t test. Source data are available for this figure.

    Journal: Life Science Alliance

    Article Title: CDCP1 promotes compensatory renal growth by integrating Src and Met signaling

    doi: 10.26508/lsa.202000832

    Figure Lengend Snippet: (A) Schematic diagram of CRISPR/Cas9-based generation of Cdcp1 -knockout mouse. Blue indicates the PAM sequence and bold letters indicate the start codon. The red arrowhead indicates the cleavage site. (B) Representative picture of wild-type (+/+), Cdcp1 heterozygous (+/−) and homozygous knockout (−/−) littermate mice at 8 wk of age. (C, D) Validation of gene knockout was performed by genotyping (C) and Western blot (D). PCR verification of deletion at first exon of Cdcp1 gene using Fw and Rev primers depicted in panel (A). Lysates from mouse kidney were subjected to immunoblotting using the indicated antibodies. (E, F) Whole body (E) and kidney (F) weight of wild-type (+/+) and Cdcp1 homozygous knockout (−/−) mice at 8 wk of age. Data information: In (E, F), the mean ratios ± SD were obtained from 10 mice per group. NS, not significantly different; unpaired two-tailed t test. Source data are available for this figure.

    Article Snippet: The following primary antibodies were used in this study: anti-CDCP1 (4115), anti-CDCP1 pY734 (9050), anti-STAT3 (9132), anti-STAT3 pY705 (9145), anti-myc-tag (2276), anti-Met pY1234/1235 (3077), anti-Met pY1349 (3121) anti-Met (3127, clone 25H2), and anti-Met (8198) antibodies were all purchased from Cell Signaling Technologies, anti-CDCP1 antibody (LC-C172540) was purchased from LSBio, anti-SFK (sc-18, clone SRC2), anti-ERα (sc-542, clone MC-20), anti-Gapdh (sc-32233, clone 6C5), and anti-MMP2 (sc-10736) antibodies were all purchased from Santa Cruz Biotechnology, anti-Src pY418 (44-660G), anti-Src pY529 (44-662G), and anti-Ki67 (14-5698-82) antibodies were all purchased from Thermo Fisher Scientific, anti-Src (OP07, clone Ab-1), anti-phosphotyrosine (05-1050, clone 4G10), and anti-MMP9 (444236) antibodies were all purchased from Millipore, anti-Fyn (610163, clone 25), anti-Lyn (610003, clone 42), and anti-Yes (610375, clone 1) were all purchased from BD Bioscience, anti-collagen IV antibody (ab6586) was purchased from Abcam, anti-laminin antibody (L9393) was purchased from Sigma-Aldrich, and anti-keratin 14 antibody (PRB-155P, clone AF64) was purchased from Covance.

    Techniques: CRISPR, Knock-Out, Sequencing, Gene Knockout, Western Blot, Two Tailed Test

    (A, B) Cdcp1 wild-type (+/+), heterozygous (+/−), and homozygous knockout (−/−) mice at 8 wk of age were subjected to UNX or sham operation. Regenerative growth of remaining kidney was analyzed 8 wk after operation and increases in remaining kidney/body weight ratios were assessed. (C) The remaining kidney was removed from UNX- or sham-operated mice, and was subjected to hematoxylin-eosin (HE) staining; arrowheads indicate glomeruli. (D, E, F) The remaining kidney after UNX was subjected to microscopic immunofluorescence analysis with specific antibodies against collagen IV (magenta), and proximal tubules were visualized with FITC-LTL (green). Scale bars: 50 μm. Thickness (E) and area (F) of proximal tubules were determined by the distance (μm, n = 100) and area (μm 2 , n = 50) between FITC-LTL-stained lumen and collagen IV-enriched basement membrane as depicted in panel (D). (G) Area (μm 2 ) of glomerulus in the remaining kidney was evaluated by using HE-staining images in panel (C) (n = 50). Data information: In (B, E, F, G), the mean ratios ± SD were obtained from 10 (B) or five mice (E, F, G) per group. * P < 0.05; ** P < 0.01; **** P < 0.0001; NS, not significantly different; two-way ANOVA.

    Journal: Life Science Alliance

    Article Title: CDCP1 promotes compensatory renal growth by integrating Src and Met signaling

    doi: 10.26508/lsa.202000832

    Figure Lengend Snippet: (A, B) Cdcp1 wild-type (+/+), heterozygous (+/−), and homozygous knockout (−/−) mice at 8 wk of age were subjected to UNX or sham operation. Regenerative growth of remaining kidney was analyzed 8 wk after operation and increases in remaining kidney/body weight ratios were assessed. (C) The remaining kidney was removed from UNX- or sham-operated mice, and was subjected to hematoxylin-eosin (HE) staining; arrowheads indicate glomeruli. (D, E, F) The remaining kidney after UNX was subjected to microscopic immunofluorescence analysis with specific antibodies against collagen IV (magenta), and proximal tubules were visualized with FITC-LTL (green). Scale bars: 50 μm. Thickness (E) and area (F) of proximal tubules were determined by the distance (μm, n = 100) and area (μm 2 , n = 50) between FITC-LTL-stained lumen and collagen IV-enriched basement membrane as depicted in panel (D). (G) Area (μm 2 ) of glomerulus in the remaining kidney was evaluated by using HE-staining images in panel (C) (n = 50). Data information: In (B, E, F, G), the mean ratios ± SD were obtained from 10 (B) or five mice (E, F, G) per group. * P < 0.05; ** P < 0.01; **** P < 0.0001; NS, not significantly different; two-way ANOVA.

    Article Snippet: The following primary antibodies were used in this study: anti-CDCP1 (4115), anti-CDCP1 pY734 (9050), anti-STAT3 (9132), anti-STAT3 pY705 (9145), anti-myc-tag (2276), anti-Met pY1234/1235 (3077), anti-Met pY1349 (3121) anti-Met (3127, clone 25H2), and anti-Met (8198) antibodies were all purchased from Cell Signaling Technologies, anti-CDCP1 antibody (LC-C172540) was purchased from LSBio, anti-SFK (sc-18, clone SRC2), anti-ERα (sc-542, clone MC-20), anti-Gapdh (sc-32233, clone 6C5), and anti-MMP2 (sc-10736) antibodies were all purchased from Santa Cruz Biotechnology, anti-Src pY418 (44-660G), anti-Src pY529 (44-662G), and anti-Ki67 (14-5698-82) antibodies were all purchased from Thermo Fisher Scientific, anti-Src (OP07, clone Ab-1), anti-phosphotyrosine (05-1050, clone 4G10), and anti-MMP9 (444236) antibodies were all purchased from Millipore, anti-Fyn (610163, clone 25), anti-Lyn (610003, clone 42), and anti-Yes (610375, clone 1) were all purchased from BD Bioscience, anti-collagen IV antibody (ab6586) was purchased from Abcam, anti-laminin antibody (L9393) was purchased from Sigma-Aldrich, and anti-keratin 14 antibody (PRB-155P, clone AF64) was purchased from Covance.

    Techniques: Knock-Out, Staining, Immunofluorescence

    (A, B, C) Related to . Cdcp1 wild-type (+/+) and homozygous knockout (−/−) mice at 8 wk of age were subjected to UNX or sham operation. After 8 wk, weight of body (A) and the remaining kidney (B) were measured, and UNX-induced percent increase in remaining kidney/body weight ratios were assessed (C). (D) Related to . Cdcp1 wild-type (+/+), heterozygous (+/−), and homozygous knockout (−/−) mice at 8 wk of age were subjected to UNX or sham operation. The remaining kidney was removed from UNX- or sham-operated mice, and was subjected to hematoxylin-eosin (HE) staining. Scale bars indicate 1,000 μm. Data information: In (A, B, C), the mean ratios ± SD were obtained from at least 13 mice per group. * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001; NS, not significantly different; two-way ANOVA (A, B); unpaired two-tailed t test (C).

    Journal: Life Science Alliance

    Article Title: CDCP1 promotes compensatory renal growth by integrating Src and Met signaling

    doi: 10.26508/lsa.202000832

    Figure Lengend Snippet: (A, B, C) Related to . Cdcp1 wild-type (+/+) and homozygous knockout (−/−) mice at 8 wk of age were subjected to UNX or sham operation. After 8 wk, weight of body (A) and the remaining kidney (B) were measured, and UNX-induced percent increase in remaining kidney/body weight ratios were assessed (C). (D) Related to . Cdcp1 wild-type (+/+), heterozygous (+/−), and homozygous knockout (−/−) mice at 8 wk of age were subjected to UNX or sham operation. The remaining kidney was removed from UNX- or sham-operated mice, and was subjected to hematoxylin-eosin (HE) staining. Scale bars indicate 1,000 μm. Data information: In (A, B, C), the mean ratios ± SD were obtained from at least 13 mice per group. * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001; NS, not significantly different; two-way ANOVA (A, B); unpaired two-tailed t test (C).

    Article Snippet: The following primary antibodies were used in this study: anti-CDCP1 (4115), anti-CDCP1 pY734 (9050), anti-STAT3 (9132), anti-STAT3 pY705 (9145), anti-myc-tag (2276), anti-Met pY1234/1235 (3077), anti-Met pY1349 (3121) anti-Met (3127, clone 25H2), and anti-Met (8198) antibodies were all purchased from Cell Signaling Technologies, anti-CDCP1 antibody (LC-C172540) was purchased from LSBio, anti-SFK (sc-18, clone SRC2), anti-ERα (sc-542, clone MC-20), anti-Gapdh (sc-32233, clone 6C5), and anti-MMP2 (sc-10736) antibodies were all purchased from Santa Cruz Biotechnology, anti-Src pY418 (44-660G), anti-Src pY529 (44-662G), and anti-Ki67 (14-5698-82) antibodies were all purchased from Thermo Fisher Scientific, anti-Src (OP07, clone Ab-1), anti-phosphotyrosine (05-1050, clone 4G10), and anti-MMP9 (444236) antibodies were all purchased from Millipore, anti-Fyn (610163, clone 25), anti-Lyn (610003, clone 42), and anti-Yes (610375, clone 1) were all purchased from BD Bioscience, anti-collagen IV antibody (ab6586) was purchased from Abcam, anti-laminin antibody (L9393) was purchased from Sigma-Aldrich, and anti-keratin 14 antibody (PRB-155P, clone AF64) was purchased from Covance.

    Techniques: Knock-Out, Staining, Two Tailed Test

    (A, B) Cdcp1 wild-type (+/+) and homozygous knockout (−/−) mice at 8 wk of age were subjected to UNX or sham operation. Compensatory growth of remaining kidney was analyzed at the indicated time points after operation and increases in remaining kidney/body weight ratios were assessed. (C, D, E, F) The remaining kidney after UNX was subjected to microscopic immunofluorescence analysis with specific antibodies against Met pY1234/1235 (C), STAT3 pY705 (D), CDCP1 pY734 (E), and Ki67 (F) and Alexa Fluor 594–conjugated secondary antibody (magenta). Proximal tubules were visualized by staining with FITC-LTL (green). Representative images were shown. Scale bars: 50 μm. (G) Ki67-positive proximal tubules (%) were estimated by calculating the ratio of Ki67-stained tubules to the total number of tubules (n > 100). (H) Schematic model of hepatocyte growth factor-induced adaptive renal regeneration. CDCP1-Src regulates the Met-STAT3 signaling leading to compensatory renal growth through induction of ECM rearrangement and cell growth/proliferation. Data information: In (A, G), the mean ratios ± SD were obtained from at least six (A) or three mice (G) per group. * P < 0.05; ** P < 0.01; **** P < 0.0001; NS, not significantly different; two-way ANOVA was calculated compared with sham-operated control or between wild-type and knockout mice.

    Journal: Life Science Alliance

    Article Title: CDCP1 promotes compensatory renal growth by integrating Src and Met signaling

    doi: 10.26508/lsa.202000832

    Figure Lengend Snippet: (A, B) Cdcp1 wild-type (+/+) and homozygous knockout (−/−) mice at 8 wk of age were subjected to UNX or sham operation. Compensatory growth of remaining kidney was analyzed at the indicated time points after operation and increases in remaining kidney/body weight ratios were assessed. (C, D, E, F) The remaining kidney after UNX was subjected to microscopic immunofluorescence analysis with specific antibodies against Met pY1234/1235 (C), STAT3 pY705 (D), CDCP1 pY734 (E), and Ki67 (F) and Alexa Fluor 594–conjugated secondary antibody (magenta). Proximal tubules were visualized by staining with FITC-LTL (green). Representative images were shown. Scale bars: 50 μm. (G) Ki67-positive proximal tubules (%) were estimated by calculating the ratio of Ki67-stained tubules to the total number of tubules (n > 100). (H) Schematic model of hepatocyte growth factor-induced adaptive renal regeneration. CDCP1-Src regulates the Met-STAT3 signaling leading to compensatory renal growth through induction of ECM rearrangement and cell growth/proliferation. Data information: In (A, G), the mean ratios ± SD were obtained from at least six (A) or three mice (G) per group. * P < 0.05; ** P < 0.01; **** P < 0.0001; NS, not significantly different; two-way ANOVA was calculated compared with sham-operated control or between wild-type and knockout mice.

    Article Snippet: The following primary antibodies were used in this study: anti-CDCP1 (4115), anti-CDCP1 pY734 (9050), anti-STAT3 (9132), anti-STAT3 pY705 (9145), anti-myc-tag (2276), anti-Met pY1234/1235 (3077), anti-Met pY1349 (3121) anti-Met (3127, clone 25H2), and anti-Met (8198) antibodies were all purchased from Cell Signaling Technologies, anti-CDCP1 antibody (LC-C172540) was purchased from LSBio, anti-SFK (sc-18, clone SRC2), anti-ERα (sc-542, clone MC-20), anti-Gapdh (sc-32233, clone 6C5), and anti-MMP2 (sc-10736) antibodies were all purchased from Santa Cruz Biotechnology, anti-Src pY418 (44-660G), anti-Src pY529 (44-662G), and anti-Ki67 (14-5698-82) antibodies were all purchased from Thermo Fisher Scientific, anti-Src (OP07, clone Ab-1), anti-phosphotyrosine (05-1050, clone 4G10), and anti-MMP9 (444236) antibodies were all purchased from Millipore, anti-Fyn (610163, clone 25), anti-Lyn (610003, clone 42), and anti-Yes (610375, clone 1) were all purchased from BD Bioscience, anti-collagen IV antibody (ab6586) was purchased from Abcam, anti-laminin antibody (L9393) was purchased from Sigma-Aldrich, and anti-keratin 14 antibody (PRB-155P, clone AF64) was purchased from Covance.

    Techniques: Knock-Out, Immunofluorescence, Staining

    (A, B, C) The remaining kidney of wild-type ( Cdcp1 +/+) mice after UNX was subjected to immunofluorescence microscopic analysis using specific antibodies against Met pY1234/1235 (A), STAT3 pY705 (B), or Src pY418 (C) and Alexa Fluor 488-conjugated secondary antibody (green). Endosome marker EEA1 was visualized using Alexa Fluor 594–conjugated secondary antibody (magenta). (D, E, F, G) The remaining kidney of Cdcp1 wild-type (+/+) and Cdcp1 homozygous knockout (−/−) mice after UNX was subjected to immunofluorescence microscopic analysis using specific antibodies against collagen IV (D), MMP2 (F), or MMP9 (G) and Alexa Fluor 594–conjugated secondary antibody (magenta). Proximal tubules were visualized by staining with FITC-LTL (green). Representative images were shown. Scale bars indicate 50 μm. (E) Relative intensity of collagen IV surrounding FITC-LTL–positive proximal tubule (n = 50) were calculated by setting the mean value for sham-operated wild-type mice to one. See also . (H) Schematic diagram of adaptive renal regeneration of wild-type (+/+) and Cdcp1 homozygous knockout (−/−) mice. Data information: In (E), the mean ratios ± SD were obtained from five mice per group. * P < 0.05; ** P < 0.01; NS, not significantly different; two-way ANOVA.

    Journal: Life Science Alliance

    Article Title: CDCP1 promotes compensatory renal growth by integrating Src and Met signaling

    doi: 10.26508/lsa.202000832

    Figure Lengend Snippet: (A, B, C) The remaining kidney of wild-type ( Cdcp1 +/+) mice after UNX was subjected to immunofluorescence microscopic analysis using specific antibodies against Met pY1234/1235 (A), STAT3 pY705 (B), or Src pY418 (C) and Alexa Fluor 488-conjugated secondary antibody (green). Endosome marker EEA1 was visualized using Alexa Fluor 594–conjugated secondary antibody (magenta). (D, E, F, G) The remaining kidney of Cdcp1 wild-type (+/+) and Cdcp1 homozygous knockout (−/−) mice after UNX was subjected to immunofluorescence microscopic analysis using specific antibodies against collagen IV (D), MMP2 (F), or MMP9 (G) and Alexa Fluor 594–conjugated secondary antibody (magenta). Proximal tubules were visualized by staining with FITC-LTL (green). Representative images were shown. Scale bars indicate 50 μm. (E) Relative intensity of collagen IV surrounding FITC-LTL–positive proximal tubule (n = 50) were calculated by setting the mean value for sham-operated wild-type mice to one. See also . (H) Schematic diagram of adaptive renal regeneration of wild-type (+/+) and Cdcp1 homozygous knockout (−/−) mice. Data information: In (E), the mean ratios ± SD were obtained from five mice per group. * P < 0.05; ** P < 0.01; NS, not significantly different; two-way ANOVA.

    Article Snippet: The following primary antibodies were used in this study: anti-CDCP1 (4115), anti-CDCP1 pY734 (9050), anti-STAT3 (9132), anti-STAT3 pY705 (9145), anti-myc-tag (2276), anti-Met pY1234/1235 (3077), anti-Met pY1349 (3121) anti-Met (3127, clone 25H2), and anti-Met (8198) antibodies were all purchased from Cell Signaling Technologies, anti-CDCP1 antibody (LC-C172540) was purchased from LSBio, anti-SFK (sc-18, clone SRC2), anti-ERα (sc-542, clone MC-20), anti-Gapdh (sc-32233, clone 6C5), and anti-MMP2 (sc-10736) antibodies were all purchased from Santa Cruz Biotechnology, anti-Src pY418 (44-660G), anti-Src pY529 (44-662G), and anti-Ki67 (14-5698-82) antibodies were all purchased from Thermo Fisher Scientific, anti-Src (OP07, clone Ab-1), anti-phosphotyrosine (05-1050, clone 4G10), and anti-MMP9 (444236) antibodies were all purchased from Millipore, anti-Fyn (610163, clone 25), anti-Lyn (610003, clone 42), and anti-Yes (610375, clone 1) were all purchased from BD Bioscience, anti-collagen IV antibody (ab6586) was purchased from Abcam, anti-laminin antibody (L9393) was purchased from Sigma-Aldrich, and anti-keratin 14 antibody (PRB-155P, clone AF64) was purchased from Covance.

    Techniques: Immunofluorescence, Marker, Knock-Out, Staining

    (A) Down-regulation of CDCP1 induces formation of a luminal structure. CDCP1-EGFP cysts embedded within the collagen matrix were incubated in the presence of Dox (1 μg/ml) for 4 d. Cysts were then incubated for an additional 5 d in the absence of Dox. (B) Contribution of mammalian target of rapamycin pathway and STAT3-MMP axis. CDCP1-EGFP cysts were pretreated with Torin1 (50 nM) or rapamycin (Rapa, 200 nM) and marimastat (Mari, 20 μM) for 2 h and then incubated with Dox (1 μg/ml) for 4 d. (C, D) Contribution of PKCδ signaling. (C) CDCP1-Y762F-EGFP cysts embedded within the collagen matrix were incubated in the presence of Dox (1 μg/ml) for 4 d. (D) CDCP1-EGFP cysts were pretreated with Gö6983 for 2 h and then incubated with Dox (1 μg/ml) for 4 d. (E, F) Analysis of hepatocyte growth factor dependency. CDCP1-EGFP cysts were pretreated with NK4 (1 μg/ml) for 2 h and then incubated with Dox (1 μg/ml) for 4 d. (E) Actin filaments were stained with Alexa Fluor 594-phalloidin (magenta). (F) Fraction of the total number of cysts counted (n > 100) with protrusions. (G) Up-regulation of CDCP1 induces an expression of cytokeratin 14 (CK14). TRE-CDCP1-EGFP-harboring cysts were incubated with Dox (1 μg/ml) for 4 d. Cytokeratin 14 was visualized with a specific antibody (magenta). Scale bars indicate 50 μm. Data information: In (F), the mean ratios ± SD were obtained from three independent experiments. NS, not significantly different; unpaired two-tailed t test.

    Journal: Life Science Alliance

    Article Title: CDCP1 promotes compensatory renal growth by integrating Src and Met signaling

    doi: 10.26508/lsa.202000832

    Figure Lengend Snippet: (A) Down-regulation of CDCP1 induces formation of a luminal structure. CDCP1-EGFP cysts embedded within the collagen matrix were incubated in the presence of Dox (1 μg/ml) for 4 d. Cysts were then incubated for an additional 5 d in the absence of Dox. (B) Contribution of mammalian target of rapamycin pathway and STAT3-MMP axis. CDCP1-EGFP cysts were pretreated with Torin1 (50 nM) or rapamycin (Rapa, 200 nM) and marimastat (Mari, 20 μM) for 2 h and then incubated with Dox (1 μg/ml) for 4 d. (C, D) Contribution of PKCδ signaling. (C) CDCP1-Y762F-EGFP cysts embedded within the collagen matrix were incubated in the presence of Dox (1 μg/ml) for 4 d. (D) CDCP1-EGFP cysts were pretreated with Gö6983 for 2 h and then incubated with Dox (1 μg/ml) for 4 d. (E, F) Analysis of hepatocyte growth factor dependency. CDCP1-EGFP cysts were pretreated with NK4 (1 μg/ml) for 2 h and then incubated with Dox (1 μg/ml) for 4 d. (E) Actin filaments were stained with Alexa Fluor 594-phalloidin (magenta). (F) Fraction of the total number of cysts counted (n > 100) with protrusions. (G) Up-regulation of CDCP1 induces an expression of cytokeratin 14 (CK14). TRE-CDCP1-EGFP-harboring cysts were incubated with Dox (1 μg/ml) for 4 d. Cytokeratin 14 was visualized with a specific antibody (magenta). Scale bars indicate 50 μm. Data information: In (F), the mean ratios ± SD were obtained from three independent experiments. NS, not significantly different; unpaired two-tailed t test.

    Article Snippet: The following primary antibodies were used in this study: anti-CDCP1 (4115), anti-CDCP1 pY734 (9050), anti-STAT3 (9132), anti-STAT3 pY705 (9145), anti-myc-tag (2276), anti-Met pY1234/1235 (3077), anti-Met pY1349 (3121) anti-Met (3127, clone 25H2), and anti-Met (8198) antibodies were all purchased from Cell Signaling Technologies, anti-CDCP1 antibody (LC-C172540) was purchased from LSBio, anti-SFK (sc-18, clone SRC2), anti-ERα (sc-542, clone MC-20), anti-Gapdh (sc-32233, clone 6C5), and anti-MMP2 (sc-10736) antibodies were all purchased from Santa Cruz Biotechnology, anti-Src pY418 (44-660G), anti-Src pY529 (44-662G), and anti-Ki67 (14-5698-82) antibodies were all purchased from Thermo Fisher Scientific, anti-Src (OP07, clone Ab-1), anti-phosphotyrosine (05-1050, clone 4G10), and anti-MMP9 (444236) antibodies were all purchased from Millipore, anti-Fyn (610163, clone 25), anti-Lyn (610003, clone 42), and anti-Yes (610375, clone 1) were all purchased from BD Bioscience, anti-collagen IV antibody (ab6586) was purchased from Abcam, anti-laminin antibody (L9393) was purchased from Sigma-Aldrich, and anti-keratin 14 antibody (PRB-155P, clone AF64) was purchased from Covance.

    Techniques: Incubation, Staining, Expressing, Two Tailed Test

    Antibodies and Dilutions Used for IHC and Western Blot Analysis

    Journal: The American Journal of Pathology

    Article Title: Src Kinase Is Biphosphorylated at Y416/Y527 and Activates the CUB-Domain Containing Protein 1/Protein Kinase C δ Pathway in a Subset of Triple-Negative Breast Cancers

    doi: 10.1016/j.ajpath.2019.10.017

    Figure Lengend Snippet: Antibodies and Dilutions Used for IHC and Western Blot Analysis

    Article Snippet: CDCP1_pY734 , Cell Signaling Technology/9050 , 1:1000 , NA , NA.

    Techniques: Western Blot

    SFK substrates CUB-domain containing protein 1 (CDCP1) and protein kinase C δ (PKCδ) are phosphorylated in SFK_pY416 positive triple-negative breast cancer (TNBC). A: Representative patient sample with diffuse immunohistochemical staining of tumor cells for SFK_pY416, CDCP1_pY743, and PKCä_pY311. Staining for each marker was detected primarily on the cell membrane. Negative cells are predominantly stromal lymphocytes. B: Representative patient sample with a focal/low (<20%) pattern of staining for SFK_pY416, CDCP1_pY743, and PKCä_pY311 in tumor cells. C: Representative patient sample with negative staining. Insets: Areas of interest at higher magnification to show detail. D: Summary of staining results in panel of 56 TNBC samples. Main images were acquired using a 10× objective, and insets were acquired using a 40× objective. Scale bars = 100 μm (A–C). FOXA, forkhead box protein.

    Journal: The American Journal of Pathology

    Article Title: Src Kinase Is Biphosphorylated at Y416/Y527 and Activates the CUB-Domain Containing Protein 1/Protein Kinase C δ Pathway in a Subset of Triple-Negative Breast Cancers

    doi: 10.1016/j.ajpath.2019.10.017

    Figure Lengend Snippet: SFK substrates CUB-domain containing protein 1 (CDCP1) and protein kinase C δ (PKCδ) are phosphorylated in SFK_pY416 positive triple-negative breast cancer (TNBC). A: Representative patient sample with diffuse immunohistochemical staining of tumor cells for SFK_pY416, CDCP1_pY743, and PKCä_pY311. Staining for each marker was detected primarily on the cell membrane. Negative cells are predominantly stromal lymphocytes. B: Representative patient sample with a focal/low (<20%) pattern of staining for SFK_pY416, CDCP1_pY743, and PKCä_pY311 in tumor cells. C: Representative patient sample with negative staining. Insets: Areas of interest at higher magnification to show detail. D: Summary of staining results in panel of 56 TNBC samples. Main images were acquired using a 10× objective, and insets were acquired using a 40× objective. Scale bars = 100 μm (A–C). FOXA, forkhead box protein.

    Article Snippet: CDCP1_pY734 , Cell Signaling Technology/9050 , 1:1000 , NA , NA.

    Techniques: Immunohistochemical staining, Staining, Marker, Negative Staining

    Clinical and Pathologic Features of TNBC Patient Samples in Relation to High (> 20%) SFK_pY416 Expression

    Journal: The American Journal of Pathology

    Article Title: Src Kinase Is Biphosphorylated at Y416/Y527 and Activates the CUB-Domain Containing Protein 1/Protein Kinase C δ Pathway in a Subset of Triple-Negative Breast Cancers

    doi: 10.1016/j.ajpath.2019.10.017

    Figure Lengend Snippet: Clinical and Pathologic Features of TNBC Patient Samples in Relation to High (> 20%) SFK_pY416 Expression

    Article Snippet: CDCP1_pY734 , Cell Signaling Technology/9050 , 1:1000 , NA , NA.

    Techniques:

    SFK_pY416 and SFK_pY527 are co-expressed in triple-negative breast cancer (TNBC). A: Western blot analysis confirms the co-expression of phosphorylated SFK/CUB-domain containing protein 1 (CDCP1) proteins, as determined by immunohistochemistry (IHC), and shows the co-expression of SFK_pY416 and SFK_pY527. Paraffin sections of TNBC were extracted and analyzed by standard Western blot analysis technique. forkhead box protein 1 (FOXA1), vimentin, and SFK_pY416 expression levels in tumor cells, as determined by IHC, are summarized on top. Probing with SFK_pY416 antibody detects a 60-kDa band in all patient samples that were SFK_pY416+ by IHC (lanes 4 and 8 to 13) and does not detect the band in patient samples that were SFK_pY416− by IHC. Cleaved CDCP1_pY734, CDCP1_pY743, and SFK_pY527 are co-expressed with SFK_pY416. Whole cell lysate from the TNBC cell line MDA-MB-231 was used as a positive control (lane 1). TNBC-2 (lane 4) has granular cytoplasmic staining for SFK_pY416. All other SFK_pY416+ tumors had membrane staining. Equal amounts of protein were loaded to each lane, as quantitated by tryptophan fluorescence spectrometry. β-Actin was used as a loading control. B: Analysis of TNBC proteome in silico showing top eight proteins (protein names displayed next to data point) whose expression is correlated with SFK_pY416, with SFK_pY527 exhibiting the strongest correlation. TNBC tumor samples were isolated from The Cancer Proteome Atlas (TCPA) breast cancer (BRCA) data set by negativity for estrogen receptor, progesterone receptor, and human epidermal growth factor 2 receptor and analyzed for correlation of protein markers. Correlations were derived by Pearson product moment correlation test. cCDCP1, cleaved CDCP1; EGFR, epidermal growth factor receptor; FASN, fatty acid synthase; flCDCP1, full-length CDCP1; MEK1, dual specificity mitogen-activated protein kinase kinase 1; NFKBp65, nuclear factor NF-kappa-B p65 subunit; PEA15, astrocytic phosphoprotein PEA-15; RAB25, Ras-related protein rab-25; SFK, Src family kinase; SHP2, Src homology region 2–containing protein tyrosine phosphatase; STAT3, signal transducer and activator of transcription 3.

    Journal: The American Journal of Pathology

    Article Title: Src Kinase Is Biphosphorylated at Y416/Y527 and Activates the CUB-Domain Containing Protein 1/Protein Kinase C δ Pathway in a Subset of Triple-Negative Breast Cancers

    doi: 10.1016/j.ajpath.2019.10.017

    Figure Lengend Snippet: SFK_pY416 and SFK_pY527 are co-expressed in triple-negative breast cancer (TNBC). A: Western blot analysis confirms the co-expression of phosphorylated SFK/CUB-domain containing protein 1 (CDCP1) proteins, as determined by immunohistochemistry (IHC), and shows the co-expression of SFK_pY416 and SFK_pY527. Paraffin sections of TNBC were extracted and analyzed by standard Western blot analysis technique. forkhead box protein 1 (FOXA1), vimentin, and SFK_pY416 expression levels in tumor cells, as determined by IHC, are summarized on top. Probing with SFK_pY416 antibody detects a 60-kDa band in all patient samples that were SFK_pY416+ by IHC (lanes 4 and 8 to 13) and does not detect the band in patient samples that were SFK_pY416− by IHC. Cleaved CDCP1_pY734, CDCP1_pY743, and SFK_pY527 are co-expressed with SFK_pY416. Whole cell lysate from the TNBC cell line MDA-MB-231 was used as a positive control (lane 1). TNBC-2 (lane 4) has granular cytoplasmic staining for SFK_pY416. All other SFK_pY416+ tumors had membrane staining. Equal amounts of protein were loaded to each lane, as quantitated by tryptophan fluorescence spectrometry. β-Actin was used as a loading control. B: Analysis of TNBC proteome in silico showing top eight proteins (protein names displayed next to data point) whose expression is correlated with SFK_pY416, with SFK_pY527 exhibiting the strongest correlation. TNBC tumor samples were isolated from The Cancer Proteome Atlas (TCPA) breast cancer (BRCA) data set by negativity for estrogen receptor, progesterone receptor, and human epidermal growth factor 2 receptor and analyzed for correlation of protein markers. Correlations were derived by Pearson product moment correlation test. cCDCP1, cleaved CDCP1; EGFR, epidermal growth factor receptor; FASN, fatty acid synthase; flCDCP1, full-length CDCP1; MEK1, dual specificity mitogen-activated protein kinase kinase 1; NFKBp65, nuclear factor NF-kappa-B p65 subunit; PEA15, astrocytic phosphoprotein PEA-15; RAB25, Ras-related protein rab-25; SFK, Src family kinase; SHP2, Src homology region 2–containing protein tyrosine phosphatase; STAT3, signal transducer and activator of transcription 3.

    Article Snippet: CDCP1_pY734 , Cell Signaling Technology/9050 , 1:1000 , NA , NA.

    Techniques: Western Blot, Expressing, Immunohistochemistry, Positive Control, Staining, Fluorescence, In Silico, Isolation, Derivative Assay

    Western blot analysis of SFK_pY416− triple-negative breast cancer (TNBC) reveals that forkhead box protein 1 (FOXA1)+ samples tend to be SFK_pY527+ and FOXA1− samples tend to be SFK_pY527−. Paraffin sections of TNBC were extracted and analyzed by standard Western blot analysis technique. FOXA1, vimentin, and SFK_pY416 expression levels in tumor cells, as determined by immunohistochemistry (IHC), are summarized on top. A: All FOXA1+/SFK_pY416− samples tested were SFK_pY527+. The presence of SFK_pY416 in lanes 2, 4, and 6 is due to the presence of SFK_pY416+ lymphocytes in these tumor samples (shown in Supplemental Figure S1). B: Five of six FOXA1−/SFK_pY416− samples tested were SFK_pY527− (lanes 2, 3, and 5 to 7). Three FOXA1−/SFK_pY416+ tumor samples were run as controls. All samples contain phosphorylated AMP-activated protein kinase alpha (AMPKα) as a uniformly phosphorylated control for phosphoprotein extraction. Equal amounts of protein were loaded to each lane, as quantitated by tryptophan fluorescence spectrometry. β-Actin was used as a loading control. cCDCP1, cleaved CDCP1; CDCP1, CUB-domain containing protein 1; flCDCP1, full-length CDCP1; PKC, protein kinase C.

    Journal: The American Journal of Pathology

    Article Title: Src Kinase Is Biphosphorylated at Y416/Y527 and Activates the CUB-Domain Containing Protein 1/Protein Kinase C δ Pathway in a Subset of Triple-Negative Breast Cancers

    doi: 10.1016/j.ajpath.2019.10.017

    Figure Lengend Snippet: Western blot analysis of SFK_pY416− triple-negative breast cancer (TNBC) reveals that forkhead box protein 1 (FOXA1)+ samples tend to be SFK_pY527+ and FOXA1− samples tend to be SFK_pY527−. Paraffin sections of TNBC were extracted and analyzed by standard Western blot analysis technique. FOXA1, vimentin, and SFK_pY416 expression levels in tumor cells, as determined by immunohistochemistry (IHC), are summarized on top. A: All FOXA1+/SFK_pY416− samples tested were SFK_pY527+. The presence of SFK_pY416 in lanes 2, 4, and 6 is due to the presence of SFK_pY416+ lymphocytes in these tumor samples (shown in Supplemental Figure S1). B: Five of six FOXA1−/SFK_pY416− samples tested were SFK_pY527− (lanes 2, 3, and 5 to 7). Three FOXA1−/SFK_pY416+ tumor samples were run as controls. All samples contain phosphorylated AMP-activated protein kinase alpha (AMPKα) as a uniformly phosphorylated control for phosphoprotein extraction. Equal amounts of protein were loaded to each lane, as quantitated by tryptophan fluorescence spectrometry. β-Actin was used as a loading control. cCDCP1, cleaved CDCP1; CDCP1, CUB-domain containing protein 1; flCDCP1, full-length CDCP1; PKC, protein kinase C.

    Article Snippet: CDCP1_pY734 , Cell Signaling Technology/9050 , 1:1000 , NA , NA.

    Techniques: Western Blot, Expressing, Immunohistochemistry, Fluorescence

    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.

    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

    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.

    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

    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.

    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

    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.

    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

    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.

    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

    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.

    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