rabbit anti p cdcp1 y734  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc rabbit anti p cdcp1 y734
    Antibody-induced loss of <t>CDCP1</t> from the cell surface. ( A ) Western blot analysis of lysates from the indicated cell lines using two mouse monoclonal anti-CDCP1 antibodies, 10D7 and 41-2 (1 µg/ml), a commercial rabbit polyclonal anti-CDCP1 antibody, 4115 (1:2,000 dilution), and an anti-GAPDH antibody (1:10,000). ( B ) Flow cytometry analysis of the indicated cell lines for plasma membrane localized CDCP1 using 10D7 and 41-2. Fixed cells were stained with the respective anti-CDCP1 antibody followed by an APC-conjugated anti-mouse IgG, then analysed by flow cytometry. Data are displayed graphically as MFI values corrected for background signal determined from cells stained with only the APC-conjugated anti-mouse IgG. 10D7 and 41-2 identified the same proportion of CDCP1 expressing cells as: HEY 89%; CAOV3 93%; SKOV3 99%; OVTOKO 86%; OVZM6 0%; OVMZ6-CDCP1 75%. ( C, D ) Flow cytometry analysis of HEY (c) and OVMZ6-CDCP1 (d) cells treated for 30 minutes at 37°C with 10D7, 41-2 or control IgG 1 κ (5µg/ml). Treated cells were fixed and plasma membrane localized CDCP1 detected using fluorescently tagged anti-CDCP1 antibody CD318 -PE . Background signal was assessed by staining treated cells with fluorescently tagged control IgG (IgG -PE ). Data are displayed as MFI values. All data are mean ± SEM from three independent experiments. ***P<0.001.
    Rabbit Anti P Cdcp1 Y734, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/rabbit anti p cdcp1 y734/product/Cell Signaling Technology Inc
    Average 94 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    rabbit anti p cdcp1 y734 - by Bioz Stars, 2023-02
    94/100 stars

    Images

    1) Product Images from "Anti-CDCP1 immuno-conjugates for detection and inhibition of ovarian cancer"

    Article Title: Anti-CDCP1 immuno-conjugates for detection and inhibition of ovarian cancer

    Journal: Theranostics

    doi: 10.7150/thno.30736

    Antibody-induced loss of CDCP1 from the cell surface. ( A ) Western blot analysis of lysates from the indicated cell lines using two mouse monoclonal anti-CDCP1 antibodies, 10D7 and 41-2 (1 µg/ml), a commercial rabbit polyclonal anti-CDCP1 antibody, 4115 (1:2,000 dilution), and an anti-GAPDH antibody (1:10,000). ( B ) Flow cytometry analysis of the indicated cell lines for plasma membrane localized CDCP1 using 10D7 and 41-2. Fixed cells were stained with the respective anti-CDCP1 antibody followed by an APC-conjugated anti-mouse IgG, then analysed by flow cytometry. Data are displayed graphically as MFI values corrected for background signal determined from cells stained with only the APC-conjugated anti-mouse IgG. 10D7 and 41-2 identified the same proportion of CDCP1 expressing cells as: HEY 89%; CAOV3 93%; SKOV3 99%; OVTOKO 86%; OVZM6 0%; OVMZ6-CDCP1 75%. ( C, D ) Flow cytometry analysis of HEY (c) and OVMZ6-CDCP1 (d) cells treated for 30 minutes at 37°C with 10D7, 41-2 or control IgG 1 κ (5µg/ml). Treated cells were fixed and plasma membrane localized CDCP1 detected using fluorescently tagged anti-CDCP1 antibody CD318 -PE . Background signal was assessed by staining treated cells with fluorescently tagged control IgG (IgG -PE ). Data are displayed as MFI values. All data are mean ± SEM from three independent experiments. ***P<0.001.
    Figure Legend Snippet: Antibody-induced loss of CDCP1 from the cell surface. ( A ) Western blot analysis of lysates from the indicated cell lines using two mouse monoclonal anti-CDCP1 antibodies, 10D7 and 41-2 (1 µg/ml), a commercial rabbit polyclonal anti-CDCP1 antibody, 4115 (1:2,000 dilution), and an anti-GAPDH antibody (1:10,000). ( B ) Flow cytometry analysis of the indicated cell lines for plasma membrane localized CDCP1 using 10D7 and 41-2. Fixed cells were stained with the respective anti-CDCP1 antibody followed by an APC-conjugated anti-mouse IgG, then analysed by flow cytometry. Data are displayed graphically as MFI values corrected for background signal determined from cells stained with only the APC-conjugated anti-mouse IgG. 10D7 and 41-2 identified the same proportion of CDCP1 expressing cells as: HEY 89%; CAOV3 93%; SKOV3 99%; OVTOKO 86%; OVZM6 0%; OVMZ6-CDCP1 75%. ( C, D ) Flow cytometry analysis of HEY (c) and OVMZ6-CDCP1 (d) cells treated for 30 minutes at 37°C with 10D7, 41-2 or control IgG 1 κ (5µg/ml). Treated cells were fixed and plasma membrane localized CDCP1 detected using fluorescently tagged anti-CDCP1 antibody CD318 -PE . Background signal was assessed by staining treated cells with fluorescently tagged control IgG (IgG -PE ). Data are displayed as MFI values. All data are mean ± SEM from three independent experiments. ***P<0.001.

    Techniques Used: Western Blot, Flow Cytometry, Staining, Expressing

    Degradation of CDCP1 induced by internalizing mAbs 41-2 and 10D7. ( A ) Western blot analysis, using anti-CDCP1 antibody 4115 (1:2,000) and an anti-GAPDH antibody (1:10,000), of lysates from HEY cells treated with isotype matched control IgG ( lef t), 10D7 (middle) or 41-2 (right) for the indicated times. ( B ) Graph of fluorescence versus time from HeLa and HeLa-CDCP1 cells treated with 10D7 pH (5µg/ml) ( left ), and graph of fluorescence signal from six EOC cell lines following treatment with 10D7 pH or 41-2 pH (5µg/ml) for 8 hours ( right ). RFU, relative fluorescence units. ( C ) Impact of lysosomal ( left ) and proteasomal ( right ) inhibition on antibody-induced degradation of CDCP1. Top panel , Anti-CDCP1 (1:2,000) and -GAPDH (1:10,000) Western blot analysis of HEY cells treated with 10D7 in the presence or absence of the lysosomal inhibitor chloroquine (CLQ; 50 µM), or the proteasomal inhibitor MG132 (20 µM) for the indicated times. Bottom panel , Graph of the ratio of CDCP1 to GAPDH signal generated from Western blot analyses of lysates from three independent assays assessing the effect of CLQ and MG132 on 10D7-induced degradation of CDCP1. All data represent mean ± SEM from three independent experiments. *P<0.05.
    Figure Legend Snippet: Degradation of CDCP1 induced by internalizing mAbs 41-2 and 10D7. ( A ) Western blot analysis, using anti-CDCP1 antibody 4115 (1:2,000) and an anti-GAPDH antibody (1:10,000), of lysates from HEY cells treated with isotype matched control IgG ( lef t), 10D7 (middle) or 41-2 (right) for the indicated times. ( B ) Graph of fluorescence versus time from HeLa and HeLa-CDCP1 cells treated with 10D7 pH (5µg/ml) ( left ), and graph of fluorescence signal from six EOC cell lines following treatment with 10D7 pH or 41-2 pH (5µg/ml) for 8 hours ( right ). RFU, relative fluorescence units. ( C ) Impact of lysosomal ( left ) and proteasomal ( right ) inhibition on antibody-induced degradation of CDCP1. Top panel , Anti-CDCP1 (1:2,000) and -GAPDH (1:10,000) Western blot analysis of HEY cells treated with 10D7 in the presence or absence of the lysosomal inhibitor chloroquine (CLQ; 50 µM), or the proteasomal inhibitor MG132 (20 µM) for the indicated times. Bottom panel , Graph of the ratio of CDCP1 to GAPDH signal generated from Western blot analyses of lysates from three independent assays assessing the effect of CLQ and MG132 on 10D7-induced degradation of CDCP1. All data represent mean ± SEM from three independent experiments. *P<0.05.

    Techniques Used: Western Blot, Fluorescence, Inhibition, Generated

    10D7 and 41-2 bind with high affinity to the ECD of CDCP1. ( A ) Schematic representation of full length CDCP1 (CDCP1 FL ) and progressively shorter carboxyl terminal truncations (CDCP1-T358, -S416, -K554, -D665). CUB domains are colored green. ( B ) 10D7 and 41-2 (1 µg/ml) Western blot analysis of conditioned media from OVMZ6 cells transiently transfected with a control vector of constructs encoding CDCP1-T358, -S416, -K554, or -D665. ( C ) Flow cytometry analysis of HEY cells incubated with: (i) 10D7-QDot 625 for 1 h, unlabelled 41-2 for 1 h then 10D7-QDot 625 for 1 h, or concurrently with 10D7-QDot 625 and 41-2 for 1 h; or (ii) CD318 -PE for 1 h, unlabelled 10D7 or 41-2 for 1 h then CD318 -PE for 1 h, or concurrently with 10D7 or 41-2 and CD318 -PE for 1 h. ( D ) Schematic of CDCP1 showing the regions to which antibodies 10D7, 41-2, CD318 and 4115 bind. ( E ) Top panels , Sensorgrams of CDCP1-ECD (concentration range 1.56 to 50 nM) binding to immobilized 10D7 (left) and 41-2 (right) depicting association (increasing signal) and dissociation (reducing signal) over time. Bottom panel , Table of kinetic parameters. k a , association rate; k d , dissociation rate; K D , affinity constant.
    Figure Legend Snippet: 10D7 and 41-2 bind with high affinity to the ECD of CDCP1. ( A ) Schematic representation of full length CDCP1 (CDCP1 FL ) and progressively shorter carboxyl terminal truncations (CDCP1-T358, -S416, -K554, -D665). CUB domains are colored green. ( B ) 10D7 and 41-2 (1 µg/ml) Western blot analysis of conditioned media from OVMZ6 cells transiently transfected with a control vector of constructs encoding CDCP1-T358, -S416, -K554, or -D665. ( C ) Flow cytometry analysis of HEY cells incubated with: (i) 10D7-QDot 625 for 1 h, unlabelled 41-2 for 1 h then 10D7-QDot 625 for 1 h, or concurrently with 10D7-QDot 625 and 41-2 for 1 h; or (ii) CD318 -PE for 1 h, unlabelled 10D7 or 41-2 for 1 h then CD318 -PE for 1 h, or concurrently with 10D7 or 41-2 and CD318 -PE for 1 h. ( D ) Schematic of CDCP1 showing the regions to which antibodies 10D7, 41-2, CD318 and 4115 bind. ( E ) Top panels , Sensorgrams of CDCP1-ECD (concentration range 1.56 to 50 nM) binding to immobilized 10D7 (left) and 41-2 (right) depicting association (increasing signal) and dissociation (reducing signal) over time. Bottom panel , Table of kinetic parameters. k a , association rate; k d , dissociation rate; K D , affinity constant.

    Techniques Used: Western Blot, Transfection, Plasmid Preparation, Construct, Flow Cytometry, Incubation, Concentration Assay, Binding Assay

    10D7-induces cell surface rapid clustering and lysosomal trafficking of CDCP1. ( A ) Live-cell confocal microscopy images of HEY-CDCP1 GFP cells treated with 10D7 pH (5µg/ ml). Internalization of CDCP1 GFP and 10D7 pH was observed at 1 frame per second for 600 s. Insets highlight green punctate CDCP1 GFP positive cellular structures at 30 s, and white cellular structures at 600s that are positive for both CDCP1 GFP and 10D7 pH . ( B ) Graph of complex formation between IgG pH and CDCP1 GFP determined as the percentage of IgG pH signal coincident with CDCP1 GFP signal using ImageJ software analysis. ( C ) Images of the plasma membrane and proximal cytoplasmic region of HEY-CDCP1 GFP cells indicating 10D7-induced clustering of CDCP1. In untreated cells CDCP1 GFP is located diffusely on the cell surface. In treated cells, arrowheads highlight rapid 10D7-induced clustering of CDCP1 GFP and its internalization. ( D ) Left panel , Overlay of CDCP1 GFP (green) and 10D7 pH (magenta) signals in HEY-CDCP1 GFP cells after 20 minutes of treatment showing co-localization of within endosomal-like structures. Middle panel , Black and white image of CDCP1 GFP signal. Right panel , Black and white image of 10D7 pH signal. ( E ) Live-cell confocal microscopy images of HEY-CDCP1 GFP cells treated with IgG7 pH (5µg/ ml). No internalization of CDCP1 GFP was observed within 300 s of treatment with IgG7 pH .
    Figure Legend Snippet: 10D7-induces cell surface rapid clustering and lysosomal trafficking of CDCP1. ( A ) Live-cell confocal microscopy images of HEY-CDCP1 GFP cells treated with 10D7 pH (5µg/ ml). Internalization of CDCP1 GFP and 10D7 pH was observed at 1 frame per second for 600 s. Insets highlight green punctate CDCP1 GFP positive cellular structures at 30 s, and white cellular structures at 600s that are positive for both CDCP1 GFP and 10D7 pH . ( B ) Graph of complex formation between IgG pH and CDCP1 GFP determined as the percentage of IgG pH signal coincident with CDCP1 GFP signal using ImageJ software analysis. ( C ) Images of the plasma membrane and proximal cytoplasmic region of HEY-CDCP1 GFP cells indicating 10D7-induced clustering of CDCP1. In untreated cells CDCP1 GFP is located diffusely on the cell surface. In treated cells, arrowheads highlight rapid 10D7-induced clustering of CDCP1 GFP and its internalization. ( D ) Left panel , Overlay of CDCP1 GFP (green) and 10D7 pH (magenta) signals in HEY-CDCP1 GFP cells after 20 minutes of treatment showing co-localization of within endosomal-like structures. Middle panel , Black and white image of CDCP1 GFP signal. Right panel , Black and white image of 10D7 pH signal. ( E ) Live-cell confocal microscopy images of HEY-CDCP1 GFP cells treated with IgG7 pH (5µg/ ml). No internalization of CDCP1 GFP was observed within 300 s of treatment with IgG7 pH .

    Techniques Used: Confocal Microscopy, Software

    CDCP1 is tyrosine phosphorylated during 10D7-induced internalization and degradation. ( A ) Lysates from HEY cells treated with 10D7 (5µg/ml) for the indicated times were examined by Western blot analysis for CDCP1, p-CDCP1-Y734, Src, p-Src-Y416, and GAPDH. Antibody dilution was 1:2,000 except the anti-GAPDH antibody which was 1:10,000. The graphs display CDCP1 and p-CDCP1-Y734 levels determined by densitometric analysis with data representing mean ± SEM from three independent experiments. ( B ) Anti-CDCP1 (1:2,000), -GFP (1:2,000) and -GAPDH (1:10,000) Western blot analysis of fractions collected by cell surface biotinylation of HEY cells expressing CDCP1 GFP , CDCP1 GFP -Y734F, -Y743F or -Y762F. ( C ) Analysis of semi-automated computer tracking of CDCP1 GFP and CDCP1 GFP -Y734F, -Y743F and -Y762F in response to 10D7. Left , representative image of CDCP1 GFP tracks that internalized in response to 10D7 pH in HEY cells. The image is an overlay onto cells of color-coded tracks (violet, tracks that moved the shortest distance; red, the tracks that moved the greatest distance). Right , Graph of distance moved over 5 min by CDCP1 GFP and CDCP1 GFP -Y734F, -Y743F and -Y762F in response to 10D7 (5 µg/ml). Data are median and range from the 100 tracks with the highest velocity in each experimental group from three independent experiments. ***P<0.001.
    Figure Legend Snippet: CDCP1 is tyrosine phosphorylated during 10D7-induced internalization and degradation. ( A ) Lysates from HEY cells treated with 10D7 (5µg/ml) for the indicated times were examined by Western blot analysis for CDCP1, p-CDCP1-Y734, Src, p-Src-Y416, and GAPDH. Antibody dilution was 1:2,000 except the anti-GAPDH antibody which was 1:10,000. The graphs display CDCP1 and p-CDCP1-Y734 levels determined by densitometric analysis with data representing mean ± SEM from three independent experiments. ( B ) Anti-CDCP1 (1:2,000), -GFP (1:2,000) and -GAPDH (1:10,000) Western blot analysis of fractions collected by cell surface biotinylation of HEY cells expressing CDCP1 GFP , CDCP1 GFP -Y734F, -Y743F or -Y762F. ( C ) Analysis of semi-automated computer tracking of CDCP1 GFP and CDCP1 GFP -Y734F, -Y743F and -Y762F in response to 10D7. Left , representative image of CDCP1 GFP tracks that internalized in response to 10D7 pH in HEY cells. The image is an overlay onto cells of color-coded tracks (violet, tracks that moved the shortest distance; red, the tracks that moved the greatest distance). Right , Graph of distance moved over 5 min by CDCP1 GFP and CDCP1 GFP -Y734F, -Y743F and -Y762F in response to 10D7 (5 µg/ml). Data are median and range from the 100 tracks with the highest velocity in each experimental group from three independent experiments. ***P<0.001.

    Techniques Used: Western Blot, Expressing

    The Src inhibitor dasatinib blocks 10D7-induced phosphorylation and internalization of CDCP1. ( A ) HEY cells, treated for 2 h with dasatinib (200 nM), were incubated with 10D7 (5µg/ ml) for the indicated times. Lysates were examined by Western blot analysis for CDCP1 (1:2,000), pCDCP1-Y734 (1:2,000) and GAPDH (1:10,000). ( B ) Live-cell confocal microscopy images, acquired at the indicated time points after antibody treatment, of HEY-CDCP1 GFP cells pre-treated with dasatinib (200 nM), then incubated with 10D7 pH . Lower panels , 10D7 pH signal. Middle panels , CDCP1 GFP signal. Upper panels , overlay of 10D7 pH and CDCP1 GFP signals. ( C ) Graph of distance moved over 5 min by CDCP1 GFP in response to 10D7 in the presence and absence of dasatinib. Data are median and range from the 100 tracks with the highest velocity in each experimental group from three independent experiments. ***P<0.001.
    Figure Legend Snippet: The Src inhibitor dasatinib blocks 10D7-induced phosphorylation and internalization of CDCP1. ( A ) HEY cells, treated for 2 h with dasatinib (200 nM), were incubated with 10D7 (5µg/ ml) for the indicated times. Lysates were examined by Western blot analysis for CDCP1 (1:2,000), pCDCP1-Y734 (1:2,000) and GAPDH (1:10,000). ( B ) Live-cell confocal microscopy images, acquired at the indicated time points after antibody treatment, of HEY-CDCP1 GFP cells pre-treated with dasatinib (200 nM), then incubated with 10D7 pH . Lower panels , 10D7 pH signal. Middle panels , CDCP1 GFP signal. Upper panels , overlay of 10D7 pH and CDCP1 GFP signals. ( C ) Graph of distance moved over 5 min by CDCP1 GFP in response to 10D7 in the presence and absence of dasatinib. Data are median and range from the 100 tracks with the highest velocity in each experimental group from three independent experiments. ***P<0.001.

    Techniques Used: Incubation, Western Blot, Confocal Microscopy

    PET-CT imaging of an EOC PDX. ( A ) Clear cell EOC PDX PH250. Left , hematoxylin and eosin stained section highlighting clear cell features at 40X with 10X magnification (inset). Right , Anti-CDCP1 immunohistochemistry (antibody 4115) highlighting strong CDCP1 expression by malignant cells with accentuation of signal on the plasma membrane at 40X and 10X magnification (inset). ( B ) Comparison of CDCP1 expression by HEY cells and PDX PH250 cells. Left , Anti-CDCP1 (antibody 4115; 1:2,000) and GAPDH (1:10,000) Western blot analysis of lysates from HEY cells, a HEY cell xenograft, and a PH250 PDX tumor. Right , Cell surface CDCP1 receptor number determined by flow cytometry of single cell suspensions of HEY cells and PDX PH250 cells. Receptor numbers per cell are indicated above the flow cytometry peaks. ( C ) Representative PET images of mice carrying subcutaneous PH250 PDX tumors on both flanks. 89 Zr-10D7 and 89 Zr-IgG1κ were injected intravenously three weeks after tumor cell inoculation, and imaging performed 144 h later. White arrowhead, tumor nodules. Yellow arrow, 89 Zr-IgG1κ signal accumulated in the spleen. ( D ) Quantitative bio-distribution analysis of 89 Zr-10D7 and 89 Zr-IgG1κ 144 h post injection (n = 4). 10D7 accumulates in tumors to a significantly higher degree than IgG1κ which accumulates in the spleen and liver. ***, P<0.001.
    Figure Legend Snippet: PET-CT imaging of an EOC PDX. ( A ) Clear cell EOC PDX PH250. Left , hematoxylin and eosin stained section highlighting clear cell features at 40X with 10X magnification (inset). Right , Anti-CDCP1 immunohistochemistry (antibody 4115) highlighting strong CDCP1 expression by malignant cells with accentuation of signal on the plasma membrane at 40X and 10X magnification (inset). ( B ) Comparison of CDCP1 expression by HEY cells and PDX PH250 cells. Left , Anti-CDCP1 (antibody 4115; 1:2,000) and GAPDH (1:10,000) Western blot analysis of lysates from HEY cells, a HEY cell xenograft, and a PH250 PDX tumor. Right , Cell surface CDCP1 receptor number determined by flow cytometry of single cell suspensions of HEY cells and PDX PH250 cells. Receptor numbers per cell are indicated above the flow cytometry peaks. ( C ) Representative PET images of mice carrying subcutaneous PH250 PDX tumors on both flanks. 89 Zr-10D7 and 89 Zr-IgG1κ were injected intravenously three weeks after tumor cell inoculation, and imaging performed 144 h later. White arrowhead, tumor nodules. Yellow arrow, 89 Zr-IgG1κ signal accumulated in the spleen. ( D ) Quantitative bio-distribution analysis of 89 Zr-10D7 and 89 Zr-IgG1κ 144 h post injection (n = 4). 10D7 accumulates in tumors to a significantly higher degree than IgG1κ which accumulates in the spleen and liver. ***, P<0.001.

    Techniques Used: Positron Emission Tomography-Computed Tomography, Imaging, Staining, Immunohistochemistry, Expressing, Western Blot, Flow Cytometry, Injection

    10D7-MMAE selectively inhibits colony formation of CDCP1 expressing but not non-expressing EOC cells. ( A ) Commassie stained gel of IgG and 10D7, and purified products from reactions of IgG and 10D7 with MMAE. ( B ) HEY cells were treated with 10D7-MMAE (5 µg/ml) for the indicated times and lysates examined by Western blot analysis for CDCP1, p-CDCP1-Y734, Src, p-Src-Y416 and GAPDH. Antibody dilution was 1:2,000 except the anti-GAPDH antibody which was 1:10,000. ( C ) Representative images of crystal violet stained colonies formed from HEY and OVMZ6-CDCP1 cells after treatment with the indicated concentrations of IgG, IgG-MMAE, 10D7 or 10D7-MMAE. ( D ) Graph of crystal violet staining, as a percentage of area (% Area), of colonies formed by HEY, OVMZ6-CDCP1 and OVMZ6 cells after treatment with increasing concentrations of IgG, IgG-MMAE, 10D7 or 10D7-MMAE. Data represent means ± SEM from three independent experiments. ***, P<0.001.
    Figure Legend Snippet: 10D7-MMAE selectively inhibits colony formation of CDCP1 expressing but not non-expressing EOC cells. ( A ) Commassie stained gel of IgG and 10D7, and purified products from reactions of IgG and 10D7 with MMAE. ( B ) HEY cells were treated with 10D7-MMAE (5 µg/ml) for the indicated times and lysates examined by Western blot analysis for CDCP1, p-CDCP1-Y734, Src, p-Src-Y416 and GAPDH. Antibody dilution was 1:2,000 except the anti-GAPDH antibody which was 1:10,000. ( C ) Representative images of crystal violet stained colonies formed from HEY and OVMZ6-CDCP1 cells after treatment with the indicated concentrations of IgG, IgG-MMAE, 10D7 or 10D7-MMAE. ( D ) Graph of crystal violet staining, as a percentage of area (% Area), of colonies formed by HEY, OVMZ6-CDCP1 and OVMZ6 cells after treatment with increasing concentrations of IgG, IgG-MMAE, 10D7 or 10D7-MMAE. Data represent means ± SEM from three independent experiments. ***, P<0.001.

    Techniques Used: Expressing, Staining, Purification, Western Blot

    rabbit anti p cdcp1 y734  (Cell Signaling Technology Inc)


    Bioz Verified Symbol Cell Signaling Technology Inc is a verified supplier
    Bioz Manufacturer Symbol Cell Signaling Technology Inc manufactures this product  
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  • 94

    Structured Review

    Cell Signaling Technology Inc rabbit anti p cdcp1 y734
    Antibody-induced loss of <t>CDCP1</t> from the cell surface. ( A ) Western blot analysis of lysates from the indicated cell lines using two mouse monoclonal anti-CDCP1 antibodies, 10D7 and 41-2 (1 µg/ml), a commercial rabbit polyclonal anti-CDCP1 antibody, 4115 (1:2,000 dilution), and an anti-GAPDH antibody (1:10,000). ( B ) Flow cytometry analysis of the indicated cell lines for plasma membrane localized CDCP1 using 10D7 and 41-2. Fixed cells were stained with the respective anti-CDCP1 antibody followed by an APC-conjugated anti-mouse IgG, then analysed by flow cytometry. Data are displayed graphically as MFI values corrected for background signal determined from cells stained with only the APC-conjugated anti-mouse IgG. 10D7 and 41-2 identified the same proportion of CDCP1 expressing cells as: HEY 89%; CAOV3 93%; SKOV3 99%; OVTOKO 86%; OVZM6 0%; OVMZ6-CDCP1 75%. ( C, D ) Flow cytometry analysis of HEY (c) and OVMZ6-CDCP1 (d) cells treated for 30 minutes at 37°C with 10D7, 41-2 or control IgG 1 κ (5µg/ml). Treated cells were fixed and plasma membrane localized CDCP1 detected using fluorescently tagged anti-CDCP1 antibody CD318 -PE . Background signal was assessed by staining treated cells with fluorescently tagged control IgG (IgG -PE ). Data are displayed as MFI values. All data are mean ± SEM from three independent experiments. ***P<0.001.
    Rabbit Anti P Cdcp1 Y734, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/rabbit anti p cdcp1 y734/product/Cell Signaling Technology Inc
    Average 94 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    rabbit anti p cdcp1 y734 - by Bioz Stars, 2023-02
    94/100 stars

    Images

    1) Product Images from "Anti-CDCP1 immuno-conjugates for detection and inhibition of ovarian cancer"

    Article Title: Anti-CDCP1 immuno-conjugates for detection and inhibition of ovarian cancer

    Journal: Theranostics

    doi: 10.7150/thno.30736

    Antibody-induced loss of CDCP1 from the cell surface. ( A ) Western blot analysis of lysates from the indicated cell lines using two mouse monoclonal anti-CDCP1 antibodies, 10D7 and 41-2 (1 µg/ml), a commercial rabbit polyclonal anti-CDCP1 antibody, 4115 (1:2,000 dilution), and an anti-GAPDH antibody (1:10,000). ( B ) Flow cytometry analysis of the indicated cell lines for plasma membrane localized CDCP1 using 10D7 and 41-2. Fixed cells were stained with the respective anti-CDCP1 antibody followed by an APC-conjugated anti-mouse IgG, then analysed by flow cytometry. Data are displayed graphically as MFI values corrected for background signal determined from cells stained with only the APC-conjugated anti-mouse IgG. 10D7 and 41-2 identified the same proportion of CDCP1 expressing cells as: HEY 89%; CAOV3 93%; SKOV3 99%; OVTOKO 86%; OVZM6 0%; OVMZ6-CDCP1 75%. ( C, D ) Flow cytometry analysis of HEY (c) and OVMZ6-CDCP1 (d) cells treated for 30 minutes at 37°C with 10D7, 41-2 or control IgG 1 κ (5µg/ml). Treated cells were fixed and plasma membrane localized CDCP1 detected using fluorescently tagged anti-CDCP1 antibody CD318 -PE . Background signal was assessed by staining treated cells with fluorescently tagged control IgG (IgG -PE ). Data are displayed as MFI values. All data are mean ± SEM from three independent experiments. ***P<0.001.
    Figure Legend Snippet: Antibody-induced loss of CDCP1 from the cell surface. ( A ) Western blot analysis of lysates from the indicated cell lines using two mouse monoclonal anti-CDCP1 antibodies, 10D7 and 41-2 (1 µg/ml), a commercial rabbit polyclonal anti-CDCP1 antibody, 4115 (1:2,000 dilution), and an anti-GAPDH antibody (1:10,000). ( B ) Flow cytometry analysis of the indicated cell lines for plasma membrane localized CDCP1 using 10D7 and 41-2. Fixed cells were stained with the respective anti-CDCP1 antibody followed by an APC-conjugated anti-mouse IgG, then analysed by flow cytometry. Data are displayed graphically as MFI values corrected for background signal determined from cells stained with only the APC-conjugated anti-mouse IgG. 10D7 and 41-2 identified the same proportion of CDCP1 expressing cells as: HEY 89%; CAOV3 93%; SKOV3 99%; OVTOKO 86%; OVZM6 0%; OVMZ6-CDCP1 75%. ( C, D ) Flow cytometry analysis of HEY (c) and OVMZ6-CDCP1 (d) cells treated for 30 minutes at 37°C with 10D7, 41-2 or control IgG 1 κ (5µg/ml). Treated cells were fixed and plasma membrane localized CDCP1 detected using fluorescently tagged anti-CDCP1 antibody CD318 -PE . Background signal was assessed by staining treated cells with fluorescently tagged control IgG (IgG -PE ). Data are displayed as MFI values. All data are mean ± SEM from three independent experiments. ***P<0.001.

    Techniques Used: Western Blot, Flow Cytometry, Staining, Expressing

    Degradation of CDCP1 induced by internalizing mAbs 41-2 and 10D7. ( A ) Western blot analysis, using anti-CDCP1 antibody 4115 (1:2,000) and an anti-GAPDH antibody (1:10,000), of lysates from HEY cells treated with isotype matched control IgG ( lef t), 10D7 (middle) or 41-2 (right) for the indicated times. ( B ) Graph of fluorescence versus time from HeLa and HeLa-CDCP1 cells treated with 10D7 pH (5µg/ml) ( left ), and graph of fluorescence signal from six EOC cell lines following treatment with 10D7 pH or 41-2 pH (5µg/ml) for 8 hours ( right ). RFU, relative fluorescence units. ( C ) Impact of lysosomal ( left ) and proteasomal ( right ) inhibition on antibody-induced degradation of CDCP1. Top panel , Anti-CDCP1 (1:2,000) and -GAPDH (1:10,000) Western blot analysis of HEY cells treated with 10D7 in the presence or absence of the lysosomal inhibitor chloroquine (CLQ; 50 µM), or the proteasomal inhibitor MG132 (20 µM) for the indicated times. Bottom panel , Graph of the ratio of CDCP1 to GAPDH signal generated from Western blot analyses of lysates from three independent assays assessing the effect of CLQ and MG132 on 10D7-induced degradation of CDCP1. All data represent mean ± SEM from three independent experiments. *P<0.05.
    Figure Legend Snippet: Degradation of CDCP1 induced by internalizing mAbs 41-2 and 10D7. ( A ) Western blot analysis, using anti-CDCP1 antibody 4115 (1:2,000) and an anti-GAPDH antibody (1:10,000), of lysates from HEY cells treated with isotype matched control IgG ( lef t), 10D7 (middle) or 41-2 (right) for the indicated times. ( B ) Graph of fluorescence versus time from HeLa and HeLa-CDCP1 cells treated with 10D7 pH (5µg/ml) ( left ), and graph of fluorescence signal from six EOC cell lines following treatment with 10D7 pH or 41-2 pH (5µg/ml) for 8 hours ( right ). RFU, relative fluorescence units. ( C ) Impact of lysosomal ( left ) and proteasomal ( right ) inhibition on antibody-induced degradation of CDCP1. Top panel , Anti-CDCP1 (1:2,000) and -GAPDH (1:10,000) Western blot analysis of HEY cells treated with 10D7 in the presence or absence of the lysosomal inhibitor chloroquine (CLQ; 50 µM), or the proteasomal inhibitor MG132 (20 µM) for the indicated times. Bottom panel , Graph of the ratio of CDCP1 to GAPDH signal generated from Western blot analyses of lysates from three independent assays assessing the effect of CLQ and MG132 on 10D7-induced degradation of CDCP1. All data represent mean ± SEM from three independent experiments. *P<0.05.

    Techniques Used: Western Blot, Fluorescence, Inhibition, Generated

    10D7 and 41-2 bind with high affinity to the ECD of CDCP1. ( A ) Schematic representation of full length CDCP1 (CDCP1 FL ) and progressively shorter carboxyl terminal truncations (CDCP1-T358, -S416, -K554, -D665). CUB domains are colored green. ( B ) 10D7 and 41-2 (1 µg/ml) Western blot analysis of conditioned media from OVMZ6 cells transiently transfected with a control vector of constructs encoding CDCP1-T358, -S416, -K554, or -D665. ( C ) Flow cytometry analysis of HEY cells incubated with: (i) 10D7-QDot 625 for 1 h, unlabelled 41-2 for 1 h then 10D7-QDot 625 for 1 h, or concurrently with 10D7-QDot 625 and 41-2 for 1 h; or (ii) CD318 -PE for 1 h, unlabelled 10D7 or 41-2 for 1 h then CD318 -PE for 1 h, or concurrently with 10D7 or 41-2 and CD318 -PE for 1 h. ( D ) Schematic of CDCP1 showing the regions to which antibodies 10D7, 41-2, CD318 and 4115 bind. ( E ) Top panels , Sensorgrams of CDCP1-ECD (concentration range 1.56 to 50 nM) binding to immobilized 10D7 (left) and 41-2 (right) depicting association (increasing signal) and dissociation (reducing signal) over time. Bottom panel , Table of kinetic parameters. k a , association rate; k d , dissociation rate; K D , affinity constant.
    Figure Legend Snippet: 10D7 and 41-2 bind with high affinity to the ECD of CDCP1. ( A ) Schematic representation of full length CDCP1 (CDCP1 FL ) and progressively shorter carboxyl terminal truncations (CDCP1-T358, -S416, -K554, -D665). CUB domains are colored green. ( B ) 10D7 and 41-2 (1 µg/ml) Western blot analysis of conditioned media from OVMZ6 cells transiently transfected with a control vector of constructs encoding CDCP1-T358, -S416, -K554, or -D665. ( C ) Flow cytometry analysis of HEY cells incubated with: (i) 10D7-QDot 625 for 1 h, unlabelled 41-2 for 1 h then 10D7-QDot 625 for 1 h, or concurrently with 10D7-QDot 625 and 41-2 for 1 h; or (ii) CD318 -PE for 1 h, unlabelled 10D7 or 41-2 for 1 h then CD318 -PE for 1 h, or concurrently with 10D7 or 41-2 and CD318 -PE for 1 h. ( D ) Schematic of CDCP1 showing the regions to which antibodies 10D7, 41-2, CD318 and 4115 bind. ( E ) Top panels , Sensorgrams of CDCP1-ECD (concentration range 1.56 to 50 nM) binding to immobilized 10D7 (left) and 41-2 (right) depicting association (increasing signal) and dissociation (reducing signal) over time. Bottom panel , Table of kinetic parameters. k a , association rate; k d , dissociation rate; K D , affinity constant.

    Techniques Used: Western Blot, Transfection, Plasmid Preparation, Construct, Flow Cytometry, Incubation, Concentration Assay, Binding Assay

    10D7-induces cell surface rapid clustering and lysosomal trafficking of CDCP1. ( A ) Live-cell confocal microscopy images of HEY-CDCP1 GFP cells treated with 10D7 pH (5µg/ ml). Internalization of CDCP1 GFP and 10D7 pH was observed at 1 frame per second for 600 s. Insets highlight green punctate CDCP1 GFP positive cellular structures at 30 s, and white cellular structures at 600s that are positive for both CDCP1 GFP and 10D7 pH . ( B ) Graph of complex formation between IgG pH and CDCP1 GFP determined as the percentage of IgG pH signal coincident with CDCP1 GFP signal using ImageJ software analysis. ( C ) Images of the plasma membrane and proximal cytoplasmic region of HEY-CDCP1 GFP cells indicating 10D7-induced clustering of CDCP1. In untreated cells CDCP1 GFP is located diffusely on the cell surface. In treated cells, arrowheads highlight rapid 10D7-induced clustering of CDCP1 GFP and its internalization. ( D ) Left panel , Overlay of CDCP1 GFP (green) and 10D7 pH (magenta) signals in HEY-CDCP1 GFP cells after 20 minutes of treatment showing co-localization of within endosomal-like structures. Middle panel , Black and white image of CDCP1 GFP signal. Right panel , Black and white image of 10D7 pH signal. ( E ) Live-cell confocal microscopy images of HEY-CDCP1 GFP cells treated with IgG7 pH (5µg/ ml). No internalization of CDCP1 GFP was observed within 300 s of treatment with IgG7 pH .
    Figure Legend Snippet: 10D7-induces cell surface rapid clustering and lysosomal trafficking of CDCP1. ( A ) Live-cell confocal microscopy images of HEY-CDCP1 GFP cells treated with 10D7 pH (5µg/ ml). Internalization of CDCP1 GFP and 10D7 pH was observed at 1 frame per second for 600 s. Insets highlight green punctate CDCP1 GFP positive cellular structures at 30 s, and white cellular structures at 600s that are positive for both CDCP1 GFP and 10D7 pH . ( B ) Graph of complex formation between IgG pH and CDCP1 GFP determined as the percentage of IgG pH signal coincident with CDCP1 GFP signal using ImageJ software analysis. ( C ) Images of the plasma membrane and proximal cytoplasmic region of HEY-CDCP1 GFP cells indicating 10D7-induced clustering of CDCP1. In untreated cells CDCP1 GFP is located diffusely on the cell surface. In treated cells, arrowheads highlight rapid 10D7-induced clustering of CDCP1 GFP and its internalization. ( D ) Left panel , Overlay of CDCP1 GFP (green) and 10D7 pH (magenta) signals in HEY-CDCP1 GFP cells after 20 minutes of treatment showing co-localization of within endosomal-like structures. Middle panel , Black and white image of CDCP1 GFP signal. Right panel , Black and white image of 10D7 pH signal. ( E ) Live-cell confocal microscopy images of HEY-CDCP1 GFP cells treated with IgG7 pH (5µg/ ml). No internalization of CDCP1 GFP was observed within 300 s of treatment with IgG7 pH .

    Techniques Used: Confocal Microscopy, Software

    CDCP1 is tyrosine phosphorylated during 10D7-induced internalization and degradation. ( A ) Lysates from HEY cells treated with 10D7 (5µg/ml) for the indicated times were examined by Western blot analysis for CDCP1, p-CDCP1-Y734, Src, p-Src-Y416, and GAPDH. Antibody dilution was 1:2,000 except the anti-GAPDH antibody which was 1:10,000. The graphs display CDCP1 and p-CDCP1-Y734 levels determined by densitometric analysis with data representing mean ± SEM from three independent experiments. ( B ) Anti-CDCP1 (1:2,000), -GFP (1:2,000) and -GAPDH (1:10,000) Western blot analysis of fractions collected by cell surface biotinylation of HEY cells expressing CDCP1 GFP , CDCP1 GFP -Y734F, -Y743F or -Y762F. ( C ) Analysis of semi-automated computer tracking of CDCP1 GFP and CDCP1 GFP -Y734F, -Y743F and -Y762F in response to 10D7. Left , representative image of CDCP1 GFP tracks that internalized in response to 10D7 pH in HEY cells. The image is an overlay onto cells of color-coded tracks (violet, tracks that moved the shortest distance; red, the tracks that moved the greatest distance). Right , Graph of distance moved over 5 min by CDCP1 GFP and CDCP1 GFP -Y734F, -Y743F and -Y762F in response to 10D7 (5 µg/ml). Data are median and range from the 100 tracks with the highest velocity in each experimental group from three independent experiments. ***P<0.001.
    Figure Legend Snippet: CDCP1 is tyrosine phosphorylated during 10D7-induced internalization and degradation. ( A ) Lysates from HEY cells treated with 10D7 (5µg/ml) for the indicated times were examined by Western blot analysis for CDCP1, p-CDCP1-Y734, Src, p-Src-Y416, and GAPDH. Antibody dilution was 1:2,000 except the anti-GAPDH antibody which was 1:10,000. The graphs display CDCP1 and p-CDCP1-Y734 levels determined by densitometric analysis with data representing mean ± SEM from three independent experiments. ( B ) Anti-CDCP1 (1:2,000), -GFP (1:2,000) and -GAPDH (1:10,000) Western blot analysis of fractions collected by cell surface biotinylation of HEY cells expressing CDCP1 GFP , CDCP1 GFP -Y734F, -Y743F or -Y762F. ( C ) Analysis of semi-automated computer tracking of CDCP1 GFP and CDCP1 GFP -Y734F, -Y743F and -Y762F in response to 10D7. Left , representative image of CDCP1 GFP tracks that internalized in response to 10D7 pH in HEY cells. The image is an overlay onto cells of color-coded tracks (violet, tracks that moved the shortest distance; red, the tracks that moved the greatest distance). Right , Graph of distance moved over 5 min by CDCP1 GFP and CDCP1 GFP -Y734F, -Y743F and -Y762F in response to 10D7 (5 µg/ml). Data are median and range from the 100 tracks with the highest velocity in each experimental group from three independent experiments. ***P<0.001.

    Techniques Used: Western Blot, Expressing

    The Src inhibitor dasatinib blocks 10D7-induced phosphorylation and internalization of CDCP1. ( A ) HEY cells, treated for 2 h with dasatinib (200 nM), were incubated with 10D7 (5µg/ ml) for the indicated times. Lysates were examined by Western blot analysis for CDCP1 (1:2,000), pCDCP1-Y734 (1:2,000) and GAPDH (1:10,000). ( B ) Live-cell confocal microscopy images, acquired at the indicated time points after antibody treatment, of HEY-CDCP1 GFP cells pre-treated with dasatinib (200 nM), then incubated with 10D7 pH . Lower panels , 10D7 pH signal. Middle panels , CDCP1 GFP signal. Upper panels , overlay of 10D7 pH and CDCP1 GFP signals. ( C ) Graph of distance moved over 5 min by CDCP1 GFP in response to 10D7 in the presence and absence of dasatinib. Data are median and range from the 100 tracks with the highest velocity in each experimental group from three independent experiments. ***P<0.001.
    Figure Legend Snippet: The Src inhibitor dasatinib blocks 10D7-induced phosphorylation and internalization of CDCP1. ( A ) HEY cells, treated for 2 h with dasatinib (200 nM), were incubated with 10D7 (5µg/ ml) for the indicated times. Lysates were examined by Western blot analysis for CDCP1 (1:2,000), pCDCP1-Y734 (1:2,000) and GAPDH (1:10,000). ( B ) Live-cell confocal microscopy images, acquired at the indicated time points after antibody treatment, of HEY-CDCP1 GFP cells pre-treated with dasatinib (200 nM), then incubated with 10D7 pH . Lower panels , 10D7 pH signal. Middle panels , CDCP1 GFP signal. Upper panels , overlay of 10D7 pH and CDCP1 GFP signals. ( C ) Graph of distance moved over 5 min by CDCP1 GFP in response to 10D7 in the presence and absence of dasatinib. Data are median and range from the 100 tracks with the highest velocity in each experimental group from three independent experiments. ***P<0.001.

    Techniques Used: Incubation, Western Blot, Confocal Microscopy

    PET-CT imaging of an EOC PDX. ( A ) Clear cell EOC PDX PH250. Left , hematoxylin and eosin stained section highlighting clear cell features at 40X with 10X magnification (inset). Right , Anti-CDCP1 immunohistochemistry (antibody 4115) highlighting strong CDCP1 expression by malignant cells with accentuation of signal on the plasma membrane at 40X and 10X magnification (inset). ( B ) Comparison of CDCP1 expression by HEY cells and PDX PH250 cells. Left , Anti-CDCP1 (antibody 4115; 1:2,000) and GAPDH (1:10,000) Western blot analysis of lysates from HEY cells, a HEY cell xenograft, and a PH250 PDX tumor. Right , Cell surface CDCP1 receptor number determined by flow cytometry of single cell suspensions of HEY cells and PDX PH250 cells. Receptor numbers per cell are indicated above the flow cytometry peaks. ( C ) Representative PET images of mice carrying subcutaneous PH250 PDX tumors on both flanks. 89 Zr-10D7 and 89 Zr-IgG1κ were injected intravenously three weeks after tumor cell inoculation, and imaging performed 144 h later. White arrowhead, tumor nodules. Yellow arrow, 89 Zr-IgG1κ signal accumulated in the spleen. ( D ) Quantitative bio-distribution analysis of 89 Zr-10D7 and 89 Zr-IgG1κ 144 h post injection (n = 4). 10D7 accumulates in tumors to a significantly higher degree than IgG1κ which accumulates in the spleen and liver. ***, P<0.001.
    Figure Legend Snippet: PET-CT imaging of an EOC PDX. ( A ) Clear cell EOC PDX PH250. Left , hematoxylin and eosin stained section highlighting clear cell features at 40X with 10X magnification (inset). Right , Anti-CDCP1 immunohistochemistry (antibody 4115) highlighting strong CDCP1 expression by malignant cells with accentuation of signal on the plasma membrane at 40X and 10X magnification (inset). ( B ) Comparison of CDCP1 expression by HEY cells and PDX PH250 cells. Left , Anti-CDCP1 (antibody 4115; 1:2,000) and GAPDH (1:10,000) Western blot analysis of lysates from HEY cells, a HEY cell xenograft, and a PH250 PDX tumor. Right , Cell surface CDCP1 receptor number determined by flow cytometry of single cell suspensions of HEY cells and PDX PH250 cells. Receptor numbers per cell are indicated above the flow cytometry peaks. ( C ) Representative PET images of mice carrying subcutaneous PH250 PDX tumors on both flanks. 89 Zr-10D7 and 89 Zr-IgG1κ were injected intravenously three weeks after tumor cell inoculation, and imaging performed 144 h later. White arrowhead, tumor nodules. Yellow arrow, 89 Zr-IgG1κ signal accumulated in the spleen. ( D ) Quantitative bio-distribution analysis of 89 Zr-10D7 and 89 Zr-IgG1κ 144 h post injection (n = 4). 10D7 accumulates in tumors to a significantly higher degree than IgG1κ which accumulates in the spleen and liver. ***, P<0.001.

    Techniques Used: Positron Emission Tomography-Computed Tomography, Imaging, Staining, Immunohistochemistry, Expressing, Western Blot, Flow Cytometry, Injection

    10D7-MMAE selectively inhibits colony formation of CDCP1 expressing but not non-expressing EOC cells. ( A ) Commassie stained gel of IgG and 10D7, and purified products from reactions of IgG and 10D7 with MMAE. ( B ) HEY cells were treated with 10D7-MMAE (5 µg/ml) for the indicated times and lysates examined by Western blot analysis for CDCP1, p-CDCP1-Y734, Src, p-Src-Y416 and GAPDH. Antibody dilution was 1:2,000 except the anti-GAPDH antibody which was 1:10,000. ( C ) Representative images of crystal violet stained colonies formed from HEY and OVMZ6-CDCP1 cells after treatment with the indicated concentrations of IgG, IgG-MMAE, 10D7 or 10D7-MMAE. ( D ) Graph of crystal violet staining, as a percentage of area (% Area), of colonies formed by HEY, OVMZ6-CDCP1 and OVMZ6 cells after treatment with increasing concentrations of IgG, IgG-MMAE, 10D7 or 10D7-MMAE. Data represent means ± SEM from three independent experiments. ***, P<0.001.
    Figure Legend Snippet: 10D7-MMAE selectively inhibits colony formation of CDCP1 expressing but not non-expressing EOC cells. ( A ) Commassie stained gel of IgG and 10D7, and purified products from reactions of IgG and 10D7 with MMAE. ( B ) HEY cells were treated with 10D7-MMAE (5 µg/ml) for the indicated times and lysates examined by Western blot analysis for CDCP1, p-CDCP1-Y734, Src, p-Src-Y416 and GAPDH. Antibody dilution was 1:2,000 except the anti-GAPDH antibody which was 1:10,000. ( C ) Representative images of crystal violet stained colonies formed from HEY and OVMZ6-CDCP1 cells after treatment with the indicated concentrations of IgG, IgG-MMAE, 10D7 or 10D7-MMAE. ( D ) Graph of crystal violet staining, as a percentage of area (% Area), of colonies formed by HEY, OVMZ6-CDCP1 and OVMZ6 cells after treatment with increasing concentrations of IgG, IgG-MMAE, 10D7 or 10D7-MMAE. Data represent means ± SEM from three independent experiments. ***, P<0.001.

    Techniques Used: Expressing, Staining, Purification, Western Blot

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    Structured Review

    Cell Signaling Technology Inc phospho cdcp1
    ( A ) Design of a PreScission Protease (Px)-cleavable <t>CDCP1</t> ectodomain fused to a TEV-releasable Fc domain with C-terminal Avi-tag (CDCP1(Px)-Fc). The R368/K369 cleavage site was replaced with a Px recognition sequence (GS) 5 -LEVLFQGP-(GS) 5 . ( B ) SDS-PAGE of CDCP1 constructs. Px treatment cleaves CDCP1(Px)-Fc into NTF and CTF-Fc fragments. NTF is heavily glycosylated (predicted 14 N-linked glycosylation sites) and runs as a smeared higher-molecular weight band at ∼60 kDa. ( C ) SEC traces of CDCP1(R368/K369A)-Fc and CDCP1(Px)-Fc treated with Px, and NTF (TEV released) show that the NTF and CTF of CDCP1(Px) remain intact after proteolysis. Numbers denote fractions corresponding to the SDS-PAGE gel lanes in B . ( D ) BLI of IgG 4A06, which recognizes the NTF, shows robust binding to both Px-treated and untreated CDCP1(Px)-Fc. ( E ) Design of Px-cleavable CDCP1 with N-terminal FLAG-tag expressed on the surface of HEK293T cells. ( F ) Flow cytometry and western blot of HEK293T-wt, HEK293T-CDCP1(R368A/K369A), HEK293T-CDCP1(Px). Flow cytometry signal of anti-FLAG and IgG 4A06 remains unchanged with Px treatment. Western blot with anti-CDCP1 D1W9N, which recognizes the C-terminal intracellular region of CDCP1, confirms Px-mediated CDCP1 proteolysis at the intended molecular weight.
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    Images

    1) Product Images from "Targeting a proteolytic neo-epitope of CUB-domain containing protein 1 in RAS-driven cancer"

    Article Title: Targeting a proteolytic neo-epitope of CUB-domain containing protein 1 in RAS-driven cancer

    Journal: bioRxiv

    doi: 10.1101/2021.06.14.448427

    ( A ) Design of a PreScission Protease (Px)-cleavable CDCP1 ectodomain fused to a TEV-releasable Fc domain with C-terminal Avi-tag (CDCP1(Px)-Fc). The R368/K369 cleavage site was replaced with a Px recognition sequence (GS) 5 -LEVLFQGP-(GS) 5 . ( B ) SDS-PAGE of CDCP1 constructs. Px treatment cleaves CDCP1(Px)-Fc into NTF and CTF-Fc fragments. NTF is heavily glycosylated (predicted 14 N-linked glycosylation sites) and runs as a smeared higher-molecular weight band at ∼60 kDa. ( C ) SEC traces of CDCP1(R368/K369A)-Fc and CDCP1(Px)-Fc treated with Px, and NTF (TEV released) show that the NTF and CTF of CDCP1(Px) remain intact after proteolysis. Numbers denote fractions corresponding to the SDS-PAGE gel lanes in B . ( D ) BLI of IgG 4A06, which recognizes the NTF, shows robust binding to both Px-treated and untreated CDCP1(Px)-Fc. ( E ) Design of Px-cleavable CDCP1 with N-terminal FLAG-tag expressed on the surface of HEK293T cells. ( F ) Flow cytometry and western blot of HEK293T-wt, HEK293T-CDCP1(R368A/K369A), HEK293T-CDCP1(Px). Flow cytometry signal of anti-FLAG and IgG 4A06 remains unchanged with Px treatment. Western blot with anti-CDCP1 D1W9N, which recognizes the C-terminal intracellular region of CDCP1, confirms Px-mediated CDCP1 proteolysis at the intended molecular weight.
    Figure Legend Snippet: ( A ) Design of a PreScission Protease (Px)-cleavable CDCP1 ectodomain fused to a TEV-releasable Fc domain with C-terminal Avi-tag (CDCP1(Px)-Fc). The R368/K369 cleavage site was replaced with a Px recognition sequence (GS) 5 -LEVLFQGP-(GS) 5 . ( B ) SDS-PAGE of CDCP1 constructs. Px treatment cleaves CDCP1(Px)-Fc into NTF and CTF-Fc fragments. NTF is heavily glycosylated (predicted 14 N-linked glycosylation sites) and runs as a smeared higher-molecular weight band at ∼60 kDa. ( C ) SEC traces of CDCP1(R368/K369A)-Fc and CDCP1(Px)-Fc treated with Px, and NTF (TEV released) show that the NTF and CTF of CDCP1(Px) remain intact after proteolysis. Numbers denote fractions corresponding to the SDS-PAGE gel lanes in B . ( D ) BLI of IgG 4A06, which recognizes the NTF, shows robust binding to both Px-treated and untreated CDCP1(Px)-Fc. ( E ) Design of Px-cleavable CDCP1 with N-terminal FLAG-tag expressed on the surface of HEK293T cells. ( F ) Flow cytometry and western blot of HEK293T-wt, HEK293T-CDCP1(R368A/K369A), HEK293T-CDCP1(Px). Flow cytometry signal of anti-FLAG and IgG 4A06 remains unchanged with Px treatment. Western blot with anti-CDCP1 D1W9N, which recognizes the C-terminal intracellular region of CDCP1, confirms Px-mediated CDCP1 proteolysis at the intended molecular weight.

    Techniques Used: Sequencing, SDS Page, Construct, Molecular Weight, Binding Assay, FLAG-tag, Flow Cytometry, Western Blot

    ( A ) Schematic of IP-MS strategy to identify the endogenous proteolysis sites of CDCP1 on PDAC cells. CDCP1 was IP-ed with IgG 4A06 or D1W9N Ab and digested with Glu-C, which cleaves after aspartic acid, and was analyzed by LC-MS/MS to identify peptides corresponding to proteolytic products of CDCP1. ( B ) ( top ) Western blot of PDAC cell lines expressing differential amounts of uncleaved and cleaved CDCP1. D1W9N Ab was used to detect C-terminal fragment of CDCP1. PL5 and PL45 express mostly cleaved CDCP1, while HPAC expresses mostly uncleaved CDCP1. HPNE, a non-malignant pancreatic cell line, expresses low levels of CDCP1. ( bottom ) IP-blot shows that IP with IgG 4A06 can pull-down the CTF of CDCP1. ( C ) Depiction of peptides and proteolysis sites identified in PL5, PL45, and HPAC cell lines. Peptides identified by LC-MS for each cell line is aligned to the reference sequence highlighted in light blue. Aspartic acid residues recognized by Glu-C are highlighted in red underlined text. Three proteolysis sites of CDCP1: Cut 1 (K365), Cut 2 (R368), Cut 3 (K369), are observed in PL5 and PL45 cells but not on HPAC cells, and are highlighted in blue text and blue vertical lines.
    Figure Legend Snippet: ( A ) Schematic of IP-MS strategy to identify the endogenous proteolysis sites of CDCP1 on PDAC cells. CDCP1 was IP-ed with IgG 4A06 or D1W9N Ab and digested with Glu-C, which cleaves after aspartic acid, and was analyzed by LC-MS/MS to identify peptides corresponding to proteolytic products of CDCP1. ( B ) ( top ) Western blot of PDAC cell lines expressing differential amounts of uncleaved and cleaved CDCP1. D1W9N Ab was used to detect C-terminal fragment of CDCP1. PL5 and PL45 express mostly cleaved CDCP1, while HPAC expresses mostly uncleaved CDCP1. HPNE, a non-malignant pancreatic cell line, expresses low levels of CDCP1. ( bottom ) IP-blot shows that IP with IgG 4A06 can pull-down the CTF of CDCP1. ( C ) Depiction of peptides and proteolysis sites identified in PL5, PL45, and HPAC cell lines. Peptides identified by LC-MS for each cell line is aligned to the reference sequence highlighted in light blue. Aspartic acid residues recognized by Glu-C are highlighted in red underlined text. Three proteolysis sites of CDCP1: Cut 1 (K365), Cut 2 (R368), Cut 3 (K369), are observed in PL5 and PL45 cells but not on HPAC cells, and are highlighted in blue text and blue vertical lines.

    Techniques Used: Liquid Chromatography with Mass Spectroscopy, Western Blot, Expressing, Sequencing

    ( A ) Schematic of the co-transfection strategy to generate c-CDCP1 ectodomain. The NTF and CTF are encoded on separate plasmids with an IL2 secretion sequence. ( B ) SDS-PAGE of fl-CDCP1 and c-CDCP1 (Cut 1, Cut 2, Cut 3) ectodomain show successful expression and purification. NTF is heavily glycosylated and runs as a high-molecular weight smear. ( C ) BLI of IgG 4A06 to fl- or c-CDCP1 ectodomains show that the NTF of CDCP1 is intact on both cleaved and uncleaved CDCP1. ( D ) Differential Scanning Fluorimetry (DSF) shows that fl- and c-CDCP1 have similar melting profiles and stabilities, suggesting the NTF/CTF complex does not dissociate until full unfolding of the protein. T m is reported as an average and standard deviation of two replicates. ( E ) Circular Dichroism (CD) spectra of fl- and c-CDCP1. CDCP1 has a β-sheet signature with minima ∼217 nm. The slight difference in spectral shape between fl- and c-CDCP1 indicates a subtle change in secondary structure. ( F ) SAXS-derived P(r) function of fl- and c-CDCP1 ectodomains show similar overall architecture. SAXS-derived ab initio envelopes are shown under the graph. ( G ) Radii of gyration (R g ) of fl- and c-CDCP1. ( H ) SEC-MALS chromatograms of fl- and c-CDCP1 show similar elution profiles and molecular weights corresponding to monomeric ectodomain.
    Figure Legend Snippet: ( A ) Schematic of the co-transfection strategy to generate c-CDCP1 ectodomain. The NTF and CTF are encoded on separate plasmids with an IL2 secretion sequence. ( B ) SDS-PAGE of fl-CDCP1 and c-CDCP1 (Cut 1, Cut 2, Cut 3) ectodomain show successful expression and purification. NTF is heavily glycosylated and runs as a high-molecular weight smear. ( C ) BLI of IgG 4A06 to fl- or c-CDCP1 ectodomains show that the NTF of CDCP1 is intact on both cleaved and uncleaved CDCP1. ( D ) Differential Scanning Fluorimetry (DSF) shows that fl- and c-CDCP1 have similar melting profiles and stabilities, suggesting the NTF/CTF complex does not dissociate until full unfolding of the protein. T m is reported as an average and standard deviation of two replicates. ( E ) Circular Dichroism (CD) spectra of fl- and c-CDCP1. CDCP1 has a β-sheet signature with minima ∼217 nm. The slight difference in spectral shape between fl- and c-CDCP1 indicates a subtle change in secondary structure. ( F ) SAXS-derived P(r) function of fl- and c-CDCP1 ectodomains show similar overall architecture. SAXS-derived ab initio envelopes are shown under the graph. ( G ) Radii of gyration (R g ) of fl- and c-CDCP1. ( H ) SEC-MALS chromatograms of fl- and c-CDCP1 show similar elution profiles and molecular weights corresponding to monomeric ectodomain.

    Techniques Used: Cotransfection, Sequencing, SDS Page, Expressing, Purification, Molecular Weight, Standard Deviation, Derivative Assay

    ( A ) Schematic of strategy to generate HEK293T cell lines expressing fl- or c-CDCP1. For c-CDCP1, a lentiviral vector was designed where a T2A self-cleavage sequence flanks the CTF (res 370-836) and NTF (res 30-369). For fl-CDCP1, a lentiviral vector encoding the full CDCP1 sequence (res 30-836) was designed. An IL2 signal sequence precedes each fragment. ( B ) Flow cytometry of IgG 4A06 to HEK293T fl-CDCP1 and HEK293T c-CDCP1 cell lines indicates the NTF of CDCP1 is present on the cell surface for all cell lines. ( D ) Western blot of CDCP1 and intracellular proteins associated with CDCP1 signaling. Both fl-CDCP1 and c-CDCP1 are phosphorylated and initiate downstream signaling mediated by Src and PKCδ. Phosphorylation of Y734 on CDCP1 is important for phosphorylation of other tyrosine residues and downstream signaling partners. Note, anti-phosphoY311-PKCδ appears to be cross-reactive to CDCP1-pY734. ( D ) Cell adhesion assay comparing HEK239T fl-CDCP1 and c-CDCP1 shows that overexpression of both fl- and c- CDCP1 decreases cell adhesion and is dependent on phosphorylation of intracellular tyrosine residues, specifically of Y734. **p = 0.0016, ****p < 0.0001, ns = not significant p >0.05. (unpaired t-test)
    Figure Legend Snippet: ( A ) Schematic of strategy to generate HEK293T cell lines expressing fl- or c-CDCP1. For c-CDCP1, a lentiviral vector was designed where a T2A self-cleavage sequence flanks the CTF (res 370-836) and NTF (res 30-369). For fl-CDCP1, a lentiviral vector encoding the full CDCP1 sequence (res 30-836) was designed. An IL2 signal sequence precedes each fragment. ( B ) Flow cytometry of IgG 4A06 to HEK293T fl-CDCP1 and HEK293T c-CDCP1 cell lines indicates the NTF of CDCP1 is present on the cell surface for all cell lines. ( D ) Western blot of CDCP1 and intracellular proteins associated with CDCP1 signaling. Both fl-CDCP1 and c-CDCP1 are phosphorylated and initiate downstream signaling mediated by Src and PKCδ. Phosphorylation of Y734 on CDCP1 is important for phosphorylation of other tyrosine residues and downstream signaling partners. Note, anti-phosphoY311-PKCδ appears to be cross-reactive to CDCP1-pY734. ( D ) Cell adhesion assay comparing HEK239T fl-CDCP1 and c-CDCP1 shows that overexpression of both fl- and c- CDCP1 decreases cell adhesion and is dependent on phosphorylation of intracellular tyrosine residues, specifically of Y734. **p = 0.0016, ****p < 0.0001, ns = not significant p >0.05. (unpaired t-test)

    Techniques Used: Expressing, Plasmid Preparation, Sequencing, Flow Cytometry, Western Blot, Cell Adhesion Assay, Over Expression

    ( A ) Differential phage selection strategy to identify a cleaved CDCP1-specific antibody. Fab-phage were pre-cleared with fl-CDCP1-Fc prior to positive selection with c-CDCP1-Fc. Enriched Fab-phage were characterized by phage ELISA for selective binding to c-CDCP1-Fc. ( B ) BLI show specific binding of IgG CL03 to c-CDCP1-Fc but not to fl-CDCP1-Fc. (K D = 150-840 pM, Table S1 ) ( C ) Negative-stain EM 3D reconstruction of c-CDCP1 with CL03 Fab. ( left ) 2D class averages of c-CDCP1(Cut3) + CL03 Fab in the absence and presence of anti-Fab V H H. ( right ) Different views of 3D EM map of CDCP1(Cut3) + CL03 Fab + V H H with crystal structure of Fab (green) and V H H (blue) modeled into the density. ( D ) Immunofluorescence of Alexa Fluor-488-labeled IgG CL03 ( left panels ) and IgG 4A06 ( right panels ) on HPAC, PL5, and HPNE cells. IgG CL03 specifically stains PL5 cells that express cleaved CDCP1, while IgG 4A06 stains both HPAC and PL5 cells. ( E ) Flow cytometry shows that IgG CL03 binds to cleaved CDCP1-expressing PL5 and PL45 cells but not HPAC or HPNE cells. (n = 3, data represent average and standard deviation). ( F ) ( top ) Schematic of antibody drug conjugate (ADC) cell killing assay. ( bottom ) Dose-dependent ADC-mediated cell killing with IgG CL03 was only observed against PL5 and PL45 cells that express cleaved CDCP1, and only in the presence of both the primary and secondary antibody. (**p = 0.004, ***p = 0.0018, unpaired T-test) ( G ) In vivo PET imaging of 89 Zr-labeled IgG CL03 in PDAC xenograft mice harboring PL5 tumors (n = 4).
    Figure Legend Snippet: ( A ) Differential phage selection strategy to identify a cleaved CDCP1-specific antibody. Fab-phage were pre-cleared with fl-CDCP1-Fc prior to positive selection with c-CDCP1-Fc. Enriched Fab-phage were characterized by phage ELISA for selective binding to c-CDCP1-Fc. ( B ) BLI show specific binding of IgG CL03 to c-CDCP1-Fc but not to fl-CDCP1-Fc. (K D = 150-840 pM, Table S1 ) ( C ) Negative-stain EM 3D reconstruction of c-CDCP1 with CL03 Fab. ( left ) 2D class averages of c-CDCP1(Cut3) + CL03 Fab in the absence and presence of anti-Fab V H H. ( right ) Different views of 3D EM map of CDCP1(Cut3) + CL03 Fab + V H H with crystal structure of Fab (green) and V H H (blue) modeled into the density. ( D ) Immunofluorescence of Alexa Fluor-488-labeled IgG CL03 ( left panels ) and IgG 4A06 ( right panels ) on HPAC, PL5, and HPNE cells. IgG CL03 specifically stains PL5 cells that express cleaved CDCP1, while IgG 4A06 stains both HPAC and PL5 cells. ( E ) Flow cytometry shows that IgG CL03 binds to cleaved CDCP1-expressing PL5 and PL45 cells but not HPAC or HPNE cells. (n = 3, data represent average and standard deviation). ( F ) ( top ) Schematic of antibody drug conjugate (ADC) cell killing assay. ( bottom ) Dose-dependent ADC-mediated cell killing with IgG CL03 was only observed against PL5 and PL45 cells that express cleaved CDCP1, and only in the presence of both the primary and secondary antibody. (**p = 0.004, ***p = 0.0018, unpaired T-test) ( G ) In vivo PET imaging of 89 Zr-labeled IgG CL03 in PDAC xenograft mice harboring PL5 tumors (n = 4).

    Techniques Used: Selection, Enzyme-linked Immunosorbent Assay, Binding Assay, Staining, Immunofluorescence, Labeling, Flow Cytometry, Expressing, Standard Deviation, In Vivo, Imaging

    ( A ) BLI show specific binding of IgG58 to mouse c-CDCP1-Fc, but not to fl-CDCP1-Fc. ( B ) Flow cytometry shows that IgG58 binds robustly to Fc1245 c-CDCP1, but not to Fc1245 WT cells (n = 3, error bars represent s.d.). ( C ) Dose-dependent ADC-mediated cell killing with IgG58-MMAF treatment was only observed with Fc1245 c-CDCP1 cells and not Fc1245 WT cells. (n = 2, error bars represent s.d.) (**p = 0.002, unpaired T-test) ( D ) Representative in vivo PET images of 89 Zr-IgG58 and 89 Zr-IgG12 in mice harboring subcutaneous Fc1245 c-CDCP1 tumors. Co-administration of 50X unlabeled IgG was used to examine target specificity. ( E ) Biodistribution of 89 Zr-IgG58 and 89 Zr-IgG12 in mice harboring subcutaneous Fc1245 c-CDCP1 tumors (n = 5 per arm). Both 89 Zr-IgG58 and 89 Zr-IgG12 signal decreased when 50X unlabeled IgG was administered, indicating target-specific localization (**p = 0.003, ***p = 0.0002, unpaired T-test). 89 Zr-IgG58 shows stronger signal in the tumor, while 89 Zr-IgG12 shows weaker tumor localization and more widespread normal tissue distribution (****p <0.0001, unpaired T-test). ( F ) ADC toxicity assay in non-tumor bearing mice. Mice (n = 5 per arm) were dosed weekly with 5, 10, 15 mg/kg of either IgG12-MMAF or IgG58-MMAF and body weight was monitored for treatment-associated toxicity. There was a significant difference between the treatment arms (F(5,32) = 3.11, p = 0.0002, ANOVA), with IgG58-MMAF treatment being better tolerated, with significant differences between IgG12-MMAF and IgG58-MMAF treatments at the 15 mg/kg dose (***p = 0.0068) and 10 mg/kg dose (**p = 0.0067) (Tukey’s multiple comparisons test). ( G-H ) Theranostic efficacy study of 177 Lu-IgG58. Mice (n = 5 per treatment arm, n = 8 for vehicle arm) were injected with 400 µCi of 177 Lu-IgG58 or vehicle 4 days after Fc1245-c-CDCP1 tumor implantation. For the 2-dose arm, the treatment was repeated 6 days later. Treatment with 177 Lu-IgG58 resulted in decreased tumor growth and increased survival compared to the vehicle arm (***p = 0.0008, ****p <0.0001, unpaired two-tailed T-test).
    Figure Legend Snippet: ( A ) BLI show specific binding of IgG58 to mouse c-CDCP1-Fc, but not to fl-CDCP1-Fc. ( B ) Flow cytometry shows that IgG58 binds robustly to Fc1245 c-CDCP1, but not to Fc1245 WT cells (n = 3, error bars represent s.d.). ( C ) Dose-dependent ADC-mediated cell killing with IgG58-MMAF treatment was only observed with Fc1245 c-CDCP1 cells and not Fc1245 WT cells. (n = 2, error bars represent s.d.) (**p = 0.002, unpaired T-test) ( D ) Representative in vivo PET images of 89 Zr-IgG58 and 89 Zr-IgG12 in mice harboring subcutaneous Fc1245 c-CDCP1 tumors. Co-administration of 50X unlabeled IgG was used to examine target specificity. ( E ) Biodistribution of 89 Zr-IgG58 and 89 Zr-IgG12 in mice harboring subcutaneous Fc1245 c-CDCP1 tumors (n = 5 per arm). Both 89 Zr-IgG58 and 89 Zr-IgG12 signal decreased when 50X unlabeled IgG was administered, indicating target-specific localization (**p = 0.003, ***p = 0.0002, unpaired T-test). 89 Zr-IgG58 shows stronger signal in the tumor, while 89 Zr-IgG12 shows weaker tumor localization and more widespread normal tissue distribution (****p <0.0001, unpaired T-test). ( F ) ADC toxicity assay in non-tumor bearing mice. Mice (n = 5 per arm) were dosed weekly with 5, 10, 15 mg/kg of either IgG12-MMAF or IgG58-MMAF and body weight was monitored for treatment-associated toxicity. There was a significant difference between the treatment arms (F(5,32) = 3.11, p = 0.0002, ANOVA), with IgG58-MMAF treatment being better tolerated, with significant differences between IgG12-MMAF and IgG58-MMAF treatments at the 15 mg/kg dose (***p = 0.0068) and 10 mg/kg dose (**p = 0.0067) (Tukey’s multiple comparisons test). ( G-H ) Theranostic efficacy study of 177 Lu-IgG58. Mice (n = 5 per treatment arm, n = 8 for vehicle arm) were injected with 400 µCi of 177 Lu-IgG58 or vehicle 4 days after Fc1245-c-CDCP1 tumor implantation. For the 2-dose arm, the treatment was repeated 6 days later. Treatment with 177 Lu-IgG58 resulted in decreased tumor growth and increased survival compared to the vehicle arm (***p = 0.0008, ****p <0.0001, unpaired two-tailed T-test).

    Techniques Used: Binding Assay, Flow Cytometry, In Vivo, Injection, Tumor Implantation, Two Tailed Test

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    (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.
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    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

    technology 9050  (Cell Signaling Technology Inc)


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    Structured Review

    Cell Signaling Technology Inc technology 9050
    Technology 9050, 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
    ( 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.
    Cdcp1 Py734, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    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

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    Structured Review

    Cell Signaling Technology Inc phospho cdcp1 tyr734
    PDGF-BB stimulation upregulates <t>CDCP1</t> in TNBC cells. a CDCP1 in MDA-MB-231 cells treated with various growth factors, cytokines, and chemokines for 48 h was analyzed by FACS and reported as percentage upregulation with respect to the maximum increase induced by WHFs (100%). Representative experiment. b WB analysis of CDCP1, phospho-CDCP1 (Y734), PDGFRβ, and phospho-PDGFRβ (Y751) in MDA-MB-231 cells treated with or without PDGF-BB 20 ng/ml for 48 h. The fold-change increase in phospho-CDCP1 and CDCP1, calculated by densitometry, was 1.6 and 1.9, respectively. c WB analysis of CDCP1 in MDA-MB-231 cells treated with or without PDGF-BB 20 ng/ml and/or cycloheximide (1 μM) for 24 h. Monoclonal anti-actin was used as the total protein loading control
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    Images

    1) Product Images from "The PDGFRβ/ERK1/2 pathway regulates CDCP1 expression in triple-negative breast cancer"

    Article Title: The PDGFRβ/ERK1/2 pathway regulates CDCP1 expression in triple-negative breast cancer

    Journal: BMC Cancer

    doi: 10.1186/s12885-018-4500-9

    PDGF-BB stimulation upregulates CDCP1 in TNBC cells. a CDCP1 in MDA-MB-231 cells treated with various growth factors, cytokines, and chemokines for 48 h was analyzed by FACS and reported as percentage upregulation with respect to the maximum increase induced by WHFs (100%). Representative experiment. b WB analysis of CDCP1, phospho-CDCP1 (Y734), PDGFRβ, and phospho-PDGFRβ (Y751) in MDA-MB-231 cells treated with or without PDGF-BB 20 ng/ml for 48 h. The fold-change increase in phospho-CDCP1 and CDCP1, calculated by densitometry, was 1.6 and 1.9, respectively. c WB analysis of CDCP1 in MDA-MB-231 cells treated with or without PDGF-BB 20 ng/ml and/or cycloheximide (1 μM) for 24 h. Monoclonal anti-actin was used as the total protein loading control
    Figure Legend Snippet: PDGF-BB stimulation upregulates CDCP1 in TNBC cells. a CDCP1 in MDA-MB-231 cells treated with various growth factors, cytokines, and chemokines for 48 h was analyzed by FACS and reported as percentage upregulation with respect to the maximum increase induced by WHFs (100%). Representative experiment. b WB analysis of CDCP1, phospho-CDCP1 (Y734), PDGFRβ, and phospho-PDGFRβ (Y751) in MDA-MB-231 cells treated with or without PDGF-BB 20 ng/ml for 48 h. The fold-change increase in phospho-CDCP1 and CDCP1, calculated by densitometry, was 1.6 and 1.9, respectively. c WB analysis of CDCP1 in MDA-MB-231 cells treated with or without PDGF-BB 20 ng/ml and/or cycloheximide (1 μM) for 24 h. Monoclonal anti-actin was used as the total protein loading control

    Techniques Used:

    PDGFRβ regulates CDCP1 expression in TNBC cells. a WB analysis of PDGFRβ and CDCP1 in MDA-MB-231 cells transfected with 100 nM PDGFRβ siRNA or the appropriate negative control. Cells were harvested at 48 h post-transfection. b WB analysis of CDCP1 and phosphoERK1/2 (T202/Y204) in MDA-MB-231 cells transfected with 100 nM PDGFRβ siRNA or the appropriate negative control and with or without 5% WHF in culture medium for 24 h. Dottes lines demarcate juxtaposed images originating from separate lines of the same western blot. The fold-change increase in CDCP1, calculated by densitometry, was 2.3 and 1.6, respectively. Monoclonal anti-actin was used as the total protein loading control
    Figure Legend Snippet: PDGFRβ regulates CDCP1 expression in TNBC cells. a WB analysis of PDGFRβ and CDCP1 in MDA-MB-231 cells transfected with 100 nM PDGFRβ siRNA or the appropriate negative control. Cells were harvested at 48 h post-transfection. b WB analysis of CDCP1 and phosphoERK1/2 (T202/Y204) in MDA-MB-231 cells transfected with 100 nM PDGFRβ siRNA or the appropriate negative control and with or without 5% WHF in culture medium for 24 h. Dottes lines demarcate juxtaposed images originating from separate lines of the same western blot. The fold-change increase in CDCP1, calculated by densitometry, was 2.3 and 1.6, respectively. Monoclonal anti-actin was used as the total protein loading control

    Techniques Used: Expressing, Transfection, Negative Control, Western Blot

    ERK1/2 activity regulates CDCP1 expression in TNBC cells. a WB analysis of phospho-ERK1/2 (T202/Y204) in MDA-MB-231 cells treated with or without 20 ng/ml PDGF-BB for 10 and 60 min. b WB analysis of CDCP1 and phosphoERK1/2 (T202/Y204) in MDA-MB-231 cells treated with or without the ERK1/2 inhibitor UO126 (2 μM) and stimulated with or without 20 ng/ml PDGF-BB for 24 h. c WB analysis of CDCP1 in MDA-MB 231 cells treated with or without UO126 (2 μM) and stimulated with or without 5% WHF in culture medium for 24 h. Dotted lines demarcate juxtaposed images originating from separate lines of the same western blot. d WB analysis of CDCP1, phosphoERK1/2 (T202/Y204), and ERK1/2 in SUM149, SUM159, MDA-MB468, BT-549, MDA-MB-231, and HCC1937 cells treated with or without UO126 (2 μM) under standard medium conditions for 24 h. Monoclonal anti-actin was used as the total protein loading control
    Figure Legend Snippet: ERK1/2 activity regulates CDCP1 expression in TNBC cells. a WB analysis of phospho-ERK1/2 (T202/Y204) in MDA-MB-231 cells treated with or without 20 ng/ml PDGF-BB for 10 and 60 min. b WB analysis of CDCP1 and phosphoERK1/2 (T202/Y204) in MDA-MB-231 cells treated with or without the ERK1/2 inhibitor UO126 (2 μM) and stimulated with or without 20 ng/ml PDGF-BB for 24 h. c WB analysis of CDCP1 in MDA-MB 231 cells treated with or without UO126 (2 μM) and stimulated with or without 5% WHF in culture medium for 24 h. Dotted lines demarcate juxtaposed images originating from separate lines of the same western blot. d WB analysis of CDCP1, phosphoERK1/2 (T202/Y204), and ERK1/2 in SUM149, SUM159, MDA-MB468, BT-549, MDA-MB-231, and HCC1937 cells treated with or without UO126 (2 μM) under standard medium conditions for 24 h. Monoclonal anti-actin was used as the total protein loading control

    Techniques Used: Activity Assay, Expressing, Western Blot

    Schematic representation of CDCP1 upregulation induced by PDGF-BB/PDGFRβ pathway through ERK1/2 activation. PDGFRβ dimerizes and is activated upon binding of the PDGF-BB ligand, causing the activation of the kinase domain, visualized as tyrosine phosphorylation (P) of the receptor molecules. In conjunction with dimerization and kinase activation, the receptor molecules undergoes a conformational changes, which allow a basal kinase activity, leading to full enzymatic activity directed toward downstream mediators such as ERK1/2. ERK1/2 activity is necessary for CDCP1 protein neo-synthesis, as demonstrated by the reduction of CDCP1 protein levels in presence of UO126, an inhibitor of ERK1/2
    Figure Legend Snippet: Schematic representation of CDCP1 upregulation induced by PDGF-BB/PDGFRβ pathway through ERK1/2 activation. PDGFRβ dimerizes and is activated upon binding of the PDGF-BB ligand, causing the activation of the kinase domain, visualized as tyrosine phosphorylation (P) of the receptor molecules. In conjunction with dimerization and kinase activation, the receptor molecules undergoes a conformational changes, which allow a basal kinase activity, leading to full enzymatic activity directed toward downstream mediators such as ERK1/2. ERK1/2 activity is necessary for CDCP1 protein neo-synthesis, as demonstrated by the reduction of CDCP1 protein levels in presence of UO126, an inhibitor of ERK1/2

    Techniques Used: Activation Assay, Binding Assay, Activity Assay

    Clinical characteristics of TNBC patients according to expression of PDGFRβ and  CDCP1
    Figure Legend Snippet: Clinical characteristics of TNBC patients according to expression of PDGFRβ and CDCP1

    Techniques Used: Expressing

    IHC staining of PDGFRβ and CDCP1 in TNBC. FFPE sections of TNBC specimens were analyzed by IHC for PDGFRβ and CDCP1. a Representative image of a PDGFRβ- and CDCP1-positive case, with plasma membrane staining, at 10X and 40X magnification; b Representative image of a PDGFRβ- and CDCP1-negative case, at 10X and 40X magnification
    Figure Legend Snippet: IHC staining of PDGFRβ and CDCP1 in TNBC. FFPE sections of TNBC specimens were analyzed by IHC for PDGFRβ and CDCP1. a Representative image of a PDGFRβ- and CDCP1-positive case, with plasma membrane staining, at 10X and 40X magnification; b Representative image of a PDGFRβ- and CDCP1-negative case, at 10X and 40X magnification

    Techniques Used: Immunohistochemistry, Staining

    PDGFRβ expression in tumor and vascular lacunae according to expression of CDCP1 (IHC CDCP1)
    Figure Legend Snippet: PDGFRβ expression in tumor and vascular lacunae according to expression of CDCP1 (IHC CDCP1)

    Techniques Used: Expressing

    Genetic alterations of CDCP1 in TNBC. FFPE sections of TNBC specimens were analyzed by dual-color FISH using for CDCP1 genetic alteration CDCP1/CEP3 probes on a FFPE sections of TNBC specimens FISH. a Representative image of TNBC specimen positive for CDCP1 IHC staining, showing tumor cells with > 3 red signals for CEP3 and > 3 green signals for the CDCP1 locus (polysomy); b Representative image of TNBC specimen negative for CDCP1 IHC staining, showing tumor cells with > 3 signals for CEP3 and < 3 green signals for the CDCP1 locus (deletion)
    Figure Legend Snippet: Genetic alterations of CDCP1 in TNBC. FFPE sections of TNBC specimens were analyzed by dual-color FISH using for CDCP1 genetic alteration CDCP1/CEP3 probes on a FFPE sections of TNBC specimens FISH. a Representative image of TNBC specimen positive for CDCP1 IHC staining, showing tumor cells with > 3 red signals for CEP3 and > 3 green signals for the CDCP1 locus (polysomy); b Representative image of TNBC specimen negative for CDCP1 IHC staining, showing tumor cells with > 3 signals for CEP3 and < 3 green signals for the CDCP1 locus (deletion)

    Techniques Used: Immunohistochemistry

    PDGFRβ expression in tumor according with expression of CDCP1 protein (IHC CDCP1) and CDCP1 genetic gain
    Figure Legend Snippet: PDGFRβ expression in tumor according with expression of CDCP1 protein (IHC CDCP1) and CDCP1 genetic gain

    Techniques Used: Expressing

    rabbit polyclonal anti cdcp1  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc rabbit polyclonal anti cdcp1
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    anti phospho cdcp1 tyr734  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc anti phospho cdcp1 tyr734
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    a plcb1  (Cell Signaling Technology Inc)


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    antibodies against cdcp1 tyr 734  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc antibodies against cdcp1 tyr 734
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    Cell Signaling Technology Inc rabbit anti p cdcp1 y734
    Antibody-induced loss of <t>CDCP1</t> from the cell surface. ( A ) Western blot analysis of lysates from the indicated cell lines using two mouse monoclonal anti-CDCP1 antibodies, 10D7 and 41-2 (1 µg/ml), a commercial rabbit polyclonal anti-CDCP1 antibody, 4115 (1:2,000 dilution), and an anti-GAPDH antibody (1:10,000). ( B ) Flow cytometry analysis of the indicated cell lines for plasma membrane localized CDCP1 using 10D7 and 41-2. Fixed cells were stained with the respective anti-CDCP1 antibody followed by an APC-conjugated anti-mouse IgG, then analysed by flow cytometry. Data are displayed graphically as MFI values corrected for background signal determined from cells stained with only the APC-conjugated anti-mouse IgG. 10D7 and 41-2 identified the same proportion of CDCP1 expressing cells as: HEY 89%; CAOV3 93%; SKOV3 99%; OVTOKO 86%; OVZM6 0%; OVMZ6-CDCP1 75%. ( C, D ) Flow cytometry analysis of HEY (c) and OVMZ6-CDCP1 (d) cells treated for 30 minutes at 37°C with 10D7, 41-2 or control IgG 1 κ (5µg/ml). Treated cells were fixed and plasma membrane localized CDCP1 detected using fluorescently tagged anti-CDCP1 antibody CD318 -PE . Background signal was assessed by staining treated cells with fluorescently tagged control IgG (IgG -PE ). Data are displayed as MFI values. All data are mean ± SEM from three independent experiments. ***P<0.001.
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    Cell Signaling Technology Inc phospho cdcp1
    ( A ) Design of a PreScission Protease (Px)-cleavable <t>CDCP1</t> ectodomain fused to a TEV-releasable Fc domain with C-terminal Avi-tag (CDCP1(Px)-Fc). The R368/K369 cleavage site was replaced with a Px recognition sequence (GS) 5 -LEVLFQGP-(GS) 5 . ( B ) SDS-PAGE of CDCP1 constructs. Px treatment cleaves CDCP1(Px)-Fc into NTF and CTF-Fc fragments. NTF is heavily glycosylated (predicted 14 N-linked glycosylation sites) and runs as a smeared higher-molecular weight band at ∼60 kDa. ( C ) SEC traces of CDCP1(R368/K369A)-Fc and CDCP1(Px)-Fc treated with Px, and NTF (TEV released) show that the NTF and CTF of CDCP1(Px) remain intact after proteolysis. Numbers denote fractions corresponding to the SDS-PAGE gel lanes in B . ( D ) BLI of IgG 4A06, which recognizes the NTF, shows robust binding to both Px-treated and untreated CDCP1(Px)-Fc. ( E ) Design of Px-cleavable CDCP1 with N-terminal FLAG-tag expressed on the surface of HEK293T cells. ( F ) Flow cytometry and western blot of HEK293T-wt, HEK293T-CDCP1(R368A/K369A), HEK293T-CDCP1(Px). Flow cytometry signal of anti-FLAG and IgG 4A06 remains unchanged with Px treatment. Western blot with anti-CDCP1 D1W9N, which recognizes the C-terminal intracellular region of CDCP1, confirms Px-mediated CDCP1 proteolysis at the intended molecular weight.
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    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.
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    Cell Signaling Technology Inc technology 9050
    (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.
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    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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    PDGF-BB stimulation upregulates <t>CDCP1</t> in TNBC cells. a CDCP1 in MDA-MB-231 cells treated with various growth factors, cytokines, and chemokines for 48 h was analyzed by FACS and reported as percentage upregulation with respect to the maximum increase induced by WHFs (100%). Representative experiment. b WB analysis of CDCP1, phospho-CDCP1 (Y734), PDGFRβ, and phospho-PDGFRβ (Y751) in MDA-MB-231 cells treated with or without PDGF-BB 20 ng/ml for 48 h. The fold-change increase in phospho-CDCP1 and CDCP1, calculated by densitometry, was 1.6 and 1.9, respectively. c WB analysis of CDCP1 in MDA-MB-231 cells treated with or without PDGF-BB 20 ng/ml and/or cycloheximide (1 μM) for 24 h. Monoclonal anti-actin was used as the total protein loading control
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    PDGF-BB stimulation upregulates <t>CDCP1</t> in TNBC cells. a CDCP1 in MDA-MB-231 cells treated with various growth factors, cytokines, and chemokines for 48 h was analyzed by FACS and reported as percentage upregulation with respect to the maximum increase induced by WHFs (100%). Representative experiment. b WB analysis of CDCP1, phospho-CDCP1 (Y734), PDGFRβ, and phospho-PDGFRβ (Y751) in MDA-MB-231 cells treated with or without PDGF-BB 20 ng/ml for 48 h. The fold-change increase in phospho-CDCP1 and CDCP1, calculated by densitometry, was 1.6 and 1.9, respectively. c WB analysis of CDCP1 in MDA-MB-231 cells treated with or without PDGF-BB 20 ng/ml and/or cycloheximide (1 μM) for 24 h. Monoclonal anti-actin was used as the total protein loading control
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    PDGF-BB stimulation upregulates <t>CDCP1</t> in TNBC cells. a CDCP1 in MDA-MB-231 cells treated with various growth factors, cytokines, and chemokines for 48 h was analyzed by FACS and reported as percentage upregulation with respect to the maximum increase induced by WHFs (100%). Representative experiment. b WB analysis of CDCP1, phospho-CDCP1 (Y734), PDGFRβ, and phospho-PDGFRβ (Y751) in MDA-MB-231 cells treated with or without PDGF-BB 20 ng/ml for 48 h. The fold-change increase in phospho-CDCP1 and CDCP1, calculated by densitometry, was 1.6 and 1.9, respectively. c WB analysis of CDCP1 in MDA-MB-231 cells treated with or without PDGF-BB 20 ng/ml and/or cycloheximide (1 μM) for 24 h. Monoclonal anti-actin was used as the total protein loading control
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    Image Search Results


    Antibody-induced loss of CDCP1 from the cell surface. ( A ) Western blot analysis of lysates from the indicated cell lines using two mouse monoclonal anti-CDCP1 antibodies, 10D7 and 41-2 (1 µg/ml), a commercial rabbit polyclonal anti-CDCP1 antibody, 4115 (1:2,000 dilution), and an anti-GAPDH antibody (1:10,000). ( B ) Flow cytometry analysis of the indicated cell lines for plasma membrane localized CDCP1 using 10D7 and 41-2. Fixed cells were stained with the respective anti-CDCP1 antibody followed by an APC-conjugated anti-mouse IgG, then analysed by flow cytometry. Data are displayed graphically as MFI values corrected for background signal determined from cells stained with only the APC-conjugated anti-mouse IgG. 10D7 and 41-2 identified the same proportion of CDCP1 expressing cells as: HEY 89%; CAOV3 93%; SKOV3 99%; OVTOKO 86%; OVZM6 0%; OVMZ6-CDCP1 75%. ( C, D ) Flow cytometry analysis of HEY (c) and OVMZ6-CDCP1 (d) cells treated for 30 minutes at 37°C with 10D7, 41-2 or control IgG 1 κ (5µg/ml). Treated cells were fixed and plasma membrane localized CDCP1 detected using fluorescently tagged anti-CDCP1 antibody CD318 -PE . Background signal was assessed by staining treated cells with fluorescently tagged control IgG (IgG -PE ). Data are displayed as MFI values. All data are mean ± SEM from three independent experiments. ***P<0.001.

    Journal: Theranostics

    Article Title: Anti-CDCP1 immuno-conjugates for detection and inhibition of ovarian cancer

    doi: 10.7150/thno.30736

    Figure Lengend Snippet: Antibody-induced loss of CDCP1 from the cell surface. ( A ) Western blot analysis of lysates from the indicated cell lines using two mouse monoclonal anti-CDCP1 antibodies, 10D7 and 41-2 (1 µg/ml), a commercial rabbit polyclonal anti-CDCP1 antibody, 4115 (1:2,000 dilution), and an anti-GAPDH antibody (1:10,000). ( B ) Flow cytometry analysis of the indicated cell lines for plasma membrane localized CDCP1 using 10D7 and 41-2. Fixed cells were stained with the respective anti-CDCP1 antibody followed by an APC-conjugated anti-mouse IgG, then analysed by flow cytometry. Data are displayed graphically as MFI values corrected for background signal determined from cells stained with only the APC-conjugated anti-mouse IgG. 10D7 and 41-2 identified the same proportion of CDCP1 expressing cells as: HEY 89%; CAOV3 93%; SKOV3 99%; OVTOKO 86%; OVZM6 0%; OVMZ6-CDCP1 75%. ( C, D ) Flow cytometry analysis of HEY (c) and OVMZ6-CDCP1 (d) cells treated for 30 minutes at 37°C with 10D7, 41-2 or control IgG 1 κ (5µg/ml). Treated cells were fixed and plasma membrane localized CDCP1 detected using fluorescently tagged anti-CDCP1 antibody CD318 -PE . Background signal was assessed by staining treated cells with fluorescently tagged control IgG (IgG -PE ). Data are displayed as MFI values. All data are mean ± SEM from three independent experiments. ***P<0.001.

    Article Snippet: Rabbit anti-CDCP1 (#4115), rabbit anti-p-CDCP1-Y734 (#9050), mouse anti-Src (#2110) and rabbit anti-p-Src-Y416 family (#2101) antibodies were from Cell Signaling Technology (Gold Coast, Australia).

    Techniques: Western Blot, Flow Cytometry, Staining, Expressing

    Degradation of CDCP1 induced by internalizing mAbs 41-2 and 10D7. ( A ) Western blot analysis, using anti-CDCP1 antibody 4115 (1:2,000) and an anti-GAPDH antibody (1:10,000), of lysates from HEY cells treated with isotype matched control IgG ( lef t), 10D7 (middle) or 41-2 (right) for the indicated times. ( B ) Graph of fluorescence versus time from HeLa and HeLa-CDCP1 cells treated with 10D7 pH (5µg/ml) ( left ), and graph of fluorescence signal from six EOC cell lines following treatment with 10D7 pH or 41-2 pH (5µg/ml) for 8 hours ( right ). RFU, relative fluorescence units. ( C ) Impact of lysosomal ( left ) and proteasomal ( right ) inhibition on antibody-induced degradation of CDCP1. Top panel , Anti-CDCP1 (1:2,000) and -GAPDH (1:10,000) Western blot analysis of HEY cells treated with 10D7 in the presence or absence of the lysosomal inhibitor chloroquine (CLQ; 50 µM), or the proteasomal inhibitor MG132 (20 µM) for the indicated times. Bottom panel , Graph of the ratio of CDCP1 to GAPDH signal generated from Western blot analyses of lysates from three independent assays assessing the effect of CLQ and MG132 on 10D7-induced degradation of CDCP1. All data represent mean ± SEM from three independent experiments. *P<0.05.

    Journal: Theranostics

    Article Title: Anti-CDCP1 immuno-conjugates for detection and inhibition of ovarian cancer

    doi: 10.7150/thno.30736

    Figure Lengend Snippet: Degradation of CDCP1 induced by internalizing mAbs 41-2 and 10D7. ( A ) Western blot analysis, using anti-CDCP1 antibody 4115 (1:2,000) and an anti-GAPDH antibody (1:10,000), of lysates from HEY cells treated with isotype matched control IgG ( lef t), 10D7 (middle) or 41-2 (right) for the indicated times. ( B ) Graph of fluorescence versus time from HeLa and HeLa-CDCP1 cells treated with 10D7 pH (5µg/ml) ( left ), and graph of fluorescence signal from six EOC cell lines following treatment with 10D7 pH or 41-2 pH (5µg/ml) for 8 hours ( right ). RFU, relative fluorescence units. ( C ) Impact of lysosomal ( left ) and proteasomal ( right ) inhibition on antibody-induced degradation of CDCP1. Top panel , Anti-CDCP1 (1:2,000) and -GAPDH (1:10,000) Western blot analysis of HEY cells treated with 10D7 in the presence or absence of the lysosomal inhibitor chloroquine (CLQ; 50 µM), or the proteasomal inhibitor MG132 (20 µM) for the indicated times. Bottom panel , Graph of the ratio of CDCP1 to GAPDH signal generated from Western blot analyses of lysates from three independent assays assessing the effect of CLQ and MG132 on 10D7-induced degradation of CDCP1. All data represent mean ± SEM from three independent experiments. *P<0.05.

    Article Snippet: Rabbit anti-CDCP1 (#4115), rabbit anti-p-CDCP1-Y734 (#9050), mouse anti-Src (#2110) and rabbit anti-p-Src-Y416 family (#2101) antibodies were from Cell Signaling Technology (Gold Coast, Australia).

    Techniques: Western Blot, Fluorescence, Inhibition, Generated

    10D7 and 41-2 bind with high affinity to the ECD of CDCP1. ( A ) Schematic representation of full length CDCP1 (CDCP1 FL ) and progressively shorter carboxyl terminal truncations (CDCP1-T358, -S416, -K554, -D665). CUB domains are colored green. ( B ) 10D7 and 41-2 (1 µg/ml) Western blot analysis of conditioned media from OVMZ6 cells transiently transfected with a control vector of constructs encoding CDCP1-T358, -S416, -K554, or -D665. ( C ) Flow cytometry analysis of HEY cells incubated with: (i) 10D7-QDot 625 for 1 h, unlabelled 41-2 for 1 h then 10D7-QDot 625 for 1 h, or concurrently with 10D7-QDot 625 and 41-2 for 1 h; or (ii) CD318 -PE for 1 h, unlabelled 10D7 or 41-2 for 1 h then CD318 -PE for 1 h, or concurrently with 10D7 or 41-2 and CD318 -PE for 1 h. ( D ) Schematic of CDCP1 showing the regions to which antibodies 10D7, 41-2, CD318 and 4115 bind. ( E ) Top panels , Sensorgrams of CDCP1-ECD (concentration range 1.56 to 50 nM) binding to immobilized 10D7 (left) and 41-2 (right) depicting association (increasing signal) and dissociation (reducing signal) over time. Bottom panel , Table of kinetic parameters. k a , association rate; k d , dissociation rate; K D , affinity constant.

    Journal: Theranostics

    Article Title: Anti-CDCP1 immuno-conjugates for detection and inhibition of ovarian cancer

    doi: 10.7150/thno.30736

    Figure Lengend Snippet: 10D7 and 41-2 bind with high affinity to the ECD of CDCP1. ( A ) Schematic representation of full length CDCP1 (CDCP1 FL ) and progressively shorter carboxyl terminal truncations (CDCP1-T358, -S416, -K554, -D665). CUB domains are colored green. ( B ) 10D7 and 41-2 (1 µg/ml) Western blot analysis of conditioned media from OVMZ6 cells transiently transfected with a control vector of constructs encoding CDCP1-T358, -S416, -K554, or -D665. ( C ) Flow cytometry analysis of HEY cells incubated with: (i) 10D7-QDot 625 for 1 h, unlabelled 41-2 for 1 h then 10D7-QDot 625 for 1 h, or concurrently with 10D7-QDot 625 and 41-2 for 1 h; or (ii) CD318 -PE for 1 h, unlabelled 10D7 or 41-2 for 1 h then CD318 -PE for 1 h, or concurrently with 10D7 or 41-2 and CD318 -PE for 1 h. ( D ) Schematic of CDCP1 showing the regions to which antibodies 10D7, 41-2, CD318 and 4115 bind. ( E ) Top panels , Sensorgrams of CDCP1-ECD (concentration range 1.56 to 50 nM) binding to immobilized 10D7 (left) and 41-2 (right) depicting association (increasing signal) and dissociation (reducing signal) over time. Bottom panel , Table of kinetic parameters. k a , association rate; k d , dissociation rate; K D , affinity constant.

    Article Snippet: Rabbit anti-CDCP1 (#4115), rabbit anti-p-CDCP1-Y734 (#9050), mouse anti-Src (#2110) and rabbit anti-p-Src-Y416 family (#2101) antibodies were from Cell Signaling Technology (Gold Coast, Australia).

    Techniques: Western Blot, Transfection, Plasmid Preparation, Construct, Flow Cytometry, Incubation, Concentration Assay, Binding Assay

    10D7-induces cell surface rapid clustering and lysosomal trafficking of CDCP1. ( A ) Live-cell confocal microscopy images of HEY-CDCP1 GFP cells treated with 10D7 pH (5µg/ ml). Internalization of CDCP1 GFP and 10D7 pH was observed at 1 frame per second for 600 s. Insets highlight green punctate CDCP1 GFP positive cellular structures at 30 s, and white cellular structures at 600s that are positive for both CDCP1 GFP and 10D7 pH . ( B ) Graph of complex formation between IgG pH and CDCP1 GFP determined as the percentage of IgG pH signal coincident with CDCP1 GFP signal using ImageJ software analysis. ( C ) Images of the plasma membrane and proximal cytoplasmic region of HEY-CDCP1 GFP cells indicating 10D7-induced clustering of CDCP1. In untreated cells CDCP1 GFP is located diffusely on the cell surface. In treated cells, arrowheads highlight rapid 10D7-induced clustering of CDCP1 GFP and its internalization. ( D ) Left panel , Overlay of CDCP1 GFP (green) and 10D7 pH (magenta) signals in HEY-CDCP1 GFP cells after 20 minutes of treatment showing co-localization of within endosomal-like structures. Middle panel , Black and white image of CDCP1 GFP signal. Right panel , Black and white image of 10D7 pH signal. ( E ) Live-cell confocal microscopy images of HEY-CDCP1 GFP cells treated with IgG7 pH (5µg/ ml). No internalization of CDCP1 GFP was observed within 300 s of treatment with IgG7 pH .

    Journal: Theranostics

    Article Title: Anti-CDCP1 immuno-conjugates for detection and inhibition of ovarian cancer

    doi: 10.7150/thno.30736

    Figure Lengend Snippet: 10D7-induces cell surface rapid clustering and lysosomal trafficking of CDCP1. ( A ) Live-cell confocal microscopy images of HEY-CDCP1 GFP cells treated with 10D7 pH (5µg/ ml). Internalization of CDCP1 GFP and 10D7 pH was observed at 1 frame per second for 600 s. Insets highlight green punctate CDCP1 GFP positive cellular structures at 30 s, and white cellular structures at 600s that are positive for both CDCP1 GFP and 10D7 pH . ( B ) Graph of complex formation between IgG pH and CDCP1 GFP determined as the percentage of IgG pH signal coincident with CDCP1 GFP signal using ImageJ software analysis. ( C ) Images of the plasma membrane and proximal cytoplasmic region of HEY-CDCP1 GFP cells indicating 10D7-induced clustering of CDCP1. In untreated cells CDCP1 GFP is located diffusely on the cell surface. In treated cells, arrowheads highlight rapid 10D7-induced clustering of CDCP1 GFP and its internalization. ( D ) Left panel , Overlay of CDCP1 GFP (green) and 10D7 pH (magenta) signals in HEY-CDCP1 GFP cells after 20 minutes of treatment showing co-localization of within endosomal-like structures. Middle panel , Black and white image of CDCP1 GFP signal. Right panel , Black and white image of 10D7 pH signal. ( E ) Live-cell confocal microscopy images of HEY-CDCP1 GFP cells treated with IgG7 pH (5µg/ ml). No internalization of CDCP1 GFP was observed within 300 s of treatment with IgG7 pH .

    Article Snippet: Rabbit anti-CDCP1 (#4115), rabbit anti-p-CDCP1-Y734 (#9050), mouse anti-Src (#2110) and rabbit anti-p-Src-Y416 family (#2101) antibodies were from Cell Signaling Technology (Gold Coast, Australia).

    Techniques: Confocal Microscopy, Software

    CDCP1 is tyrosine phosphorylated during 10D7-induced internalization and degradation. ( A ) Lysates from HEY cells treated with 10D7 (5µg/ml) for the indicated times were examined by Western blot analysis for CDCP1, p-CDCP1-Y734, Src, p-Src-Y416, and GAPDH. Antibody dilution was 1:2,000 except the anti-GAPDH antibody which was 1:10,000. The graphs display CDCP1 and p-CDCP1-Y734 levels determined by densitometric analysis with data representing mean ± SEM from three independent experiments. ( B ) Anti-CDCP1 (1:2,000), -GFP (1:2,000) and -GAPDH (1:10,000) Western blot analysis of fractions collected by cell surface biotinylation of HEY cells expressing CDCP1 GFP , CDCP1 GFP -Y734F, -Y743F or -Y762F. ( C ) Analysis of semi-automated computer tracking of CDCP1 GFP and CDCP1 GFP -Y734F, -Y743F and -Y762F in response to 10D7. Left , representative image of CDCP1 GFP tracks that internalized in response to 10D7 pH in HEY cells. The image is an overlay onto cells of color-coded tracks (violet, tracks that moved the shortest distance; red, the tracks that moved the greatest distance). Right , Graph of distance moved over 5 min by CDCP1 GFP and CDCP1 GFP -Y734F, -Y743F and -Y762F in response to 10D7 (5 µg/ml). Data are median and range from the 100 tracks with the highest velocity in each experimental group from three independent experiments. ***P<0.001.

    Journal: Theranostics

    Article Title: Anti-CDCP1 immuno-conjugates for detection and inhibition of ovarian cancer

    doi: 10.7150/thno.30736

    Figure Lengend Snippet: CDCP1 is tyrosine phosphorylated during 10D7-induced internalization and degradation. ( A ) Lysates from HEY cells treated with 10D7 (5µg/ml) for the indicated times were examined by Western blot analysis for CDCP1, p-CDCP1-Y734, Src, p-Src-Y416, and GAPDH. Antibody dilution was 1:2,000 except the anti-GAPDH antibody which was 1:10,000. The graphs display CDCP1 and p-CDCP1-Y734 levels determined by densitometric analysis with data representing mean ± SEM from three independent experiments. ( B ) Anti-CDCP1 (1:2,000), -GFP (1:2,000) and -GAPDH (1:10,000) Western blot analysis of fractions collected by cell surface biotinylation of HEY cells expressing CDCP1 GFP , CDCP1 GFP -Y734F, -Y743F or -Y762F. ( C ) Analysis of semi-automated computer tracking of CDCP1 GFP and CDCP1 GFP -Y734F, -Y743F and -Y762F in response to 10D7. Left , representative image of CDCP1 GFP tracks that internalized in response to 10D7 pH in HEY cells. The image is an overlay onto cells of color-coded tracks (violet, tracks that moved the shortest distance; red, the tracks that moved the greatest distance). Right , Graph of distance moved over 5 min by CDCP1 GFP and CDCP1 GFP -Y734F, -Y743F and -Y762F in response to 10D7 (5 µg/ml). Data are median and range from the 100 tracks with the highest velocity in each experimental group from three independent experiments. ***P<0.001.

    Article Snippet: Rabbit anti-CDCP1 (#4115), rabbit anti-p-CDCP1-Y734 (#9050), mouse anti-Src (#2110) and rabbit anti-p-Src-Y416 family (#2101) antibodies were from Cell Signaling Technology (Gold Coast, Australia).

    Techniques: Western Blot, Expressing

    The Src inhibitor dasatinib blocks 10D7-induced phosphorylation and internalization of CDCP1. ( A ) HEY cells, treated for 2 h with dasatinib (200 nM), were incubated with 10D7 (5µg/ ml) for the indicated times. Lysates were examined by Western blot analysis for CDCP1 (1:2,000), pCDCP1-Y734 (1:2,000) and GAPDH (1:10,000). ( B ) Live-cell confocal microscopy images, acquired at the indicated time points after antibody treatment, of HEY-CDCP1 GFP cells pre-treated with dasatinib (200 nM), then incubated with 10D7 pH . Lower panels , 10D7 pH signal. Middle panels , CDCP1 GFP signal. Upper panels , overlay of 10D7 pH and CDCP1 GFP signals. ( C ) Graph of distance moved over 5 min by CDCP1 GFP in response to 10D7 in the presence and absence of dasatinib. Data are median and range from the 100 tracks with the highest velocity in each experimental group from three independent experiments. ***P<0.001.

    Journal: Theranostics

    Article Title: Anti-CDCP1 immuno-conjugates for detection and inhibition of ovarian cancer

    doi: 10.7150/thno.30736

    Figure Lengend Snippet: The Src inhibitor dasatinib blocks 10D7-induced phosphorylation and internalization of CDCP1. ( A ) HEY cells, treated for 2 h with dasatinib (200 nM), were incubated with 10D7 (5µg/ ml) for the indicated times. Lysates were examined by Western blot analysis for CDCP1 (1:2,000), pCDCP1-Y734 (1:2,000) and GAPDH (1:10,000). ( B ) Live-cell confocal microscopy images, acquired at the indicated time points after antibody treatment, of HEY-CDCP1 GFP cells pre-treated with dasatinib (200 nM), then incubated with 10D7 pH . Lower panels , 10D7 pH signal. Middle panels , CDCP1 GFP signal. Upper panels , overlay of 10D7 pH and CDCP1 GFP signals. ( C ) Graph of distance moved over 5 min by CDCP1 GFP in response to 10D7 in the presence and absence of dasatinib. Data are median and range from the 100 tracks with the highest velocity in each experimental group from three independent experiments. ***P<0.001.

    Article Snippet: Rabbit anti-CDCP1 (#4115), rabbit anti-p-CDCP1-Y734 (#9050), mouse anti-Src (#2110) and rabbit anti-p-Src-Y416 family (#2101) antibodies were from Cell Signaling Technology (Gold Coast, Australia).

    Techniques: Incubation, Western Blot, Confocal Microscopy

    PET-CT imaging of an EOC PDX. ( A ) Clear cell EOC PDX PH250. Left , hematoxylin and eosin stained section highlighting clear cell features at 40X with 10X magnification (inset). Right , Anti-CDCP1 immunohistochemistry (antibody 4115) highlighting strong CDCP1 expression by malignant cells with accentuation of signal on the plasma membrane at 40X and 10X magnification (inset). ( B ) Comparison of CDCP1 expression by HEY cells and PDX PH250 cells. Left , Anti-CDCP1 (antibody 4115; 1:2,000) and GAPDH (1:10,000) Western blot analysis of lysates from HEY cells, a HEY cell xenograft, and a PH250 PDX tumor. Right , Cell surface CDCP1 receptor number determined by flow cytometry of single cell suspensions of HEY cells and PDX PH250 cells. Receptor numbers per cell are indicated above the flow cytometry peaks. ( C ) Representative PET images of mice carrying subcutaneous PH250 PDX tumors on both flanks. 89 Zr-10D7 and 89 Zr-IgG1κ were injected intravenously three weeks after tumor cell inoculation, and imaging performed 144 h later. White arrowhead, tumor nodules. Yellow arrow, 89 Zr-IgG1κ signal accumulated in the spleen. ( D ) Quantitative bio-distribution analysis of 89 Zr-10D7 and 89 Zr-IgG1κ 144 h post injection (n = 4). 10D7 accumulates in tumors to a significantly higher degree than IgG1κ which accumulates in the spleen and liver. ***, P<0.001.

    Journal: Theranostics

    Article Title: Anti-CDCP1 immuno-conjugates for detection and inhibition of ovarian cancer

    doi: 10.7150/thno.30736

    Figure Lengend Snippet: PET-CT imaging of an EOC PDX. ( A ) Clear cell EOC PDX PH250. Left , hematoxylin and eosin stained section highlighting clear cell features at 40X with 10X magnification (inset). Right , Anti-CDCP1 immunohistochemistry (antibody 4115) highlighting strong CDCP1 expression by malignant cells with accentuation of signal on the plasma membrane at 40X and 10X magnification (inset). ( B ) Comparison of CDCP1 expression by HEY cells and PDX PH250 cells. Left , Anti-CDCP1 (antibody 4115; 1:2,000) and GAPDH (1:10,000) Western blot analysis of lysates from HEY cells, a HEY cell xenograft, and a PH250 PDX tumor. Right , Cell surface CDCP1 receptor number determined by flow cytometry of single cell suspensions of HEY cells and PDX PH250 cells. Receptor numbers per cell are indicated above the flow cytometry peaks. ( C ) Representative PET images of mice carrying subcutaneous PH250 PDX tumors on both flanks. 89 Zr-10D7 and 89 Zr-IgG1κ were injected intravenously three weeks after tumor cell inoculation, and imaging performed 144 h later. White arrowhead, tumor nodules. Yellow arrow, 89 Zr-IgG1κ signal accumulated in the spleen. ( D ) Quantitative bio-distribution analysis of 89 Zr-10D7 and 89 Zr-IgG1κ 144 h post injection (n = 4). 10D7 accumulates in tumors to a significantly higher degree than IgG1κ which accumulates in the spleen and liver. ***, P<0.001.

    Article Snippet: Rabbit anti-CDCP1 (#4115), rabbit anti-p-CDCP1-Y734 (#9050), mouse anti-Src (#2110) and rabbit anti-p-Src-Y416 family (#2101) antibodies were from Cell Signaling Technology (Gold Coast, Australia).

    Techniques: Positron Emission Tomography-Computed Tomography, Imaging, Staining, Immunohistochemistry, Expressing, Western Blot, Flow Cytometry, Injection

    10D7-MMAE selectively inhibits colony formation of CDCP1 expressing but not non-expressing EOC cells. ( A ) Commassie stained gel of IgG and 10D7, and purified products from reactions of IgG and 10D7 with MMAE. ( B ) HEY cells were treated with 10D7-MMAE (5 µg/ml) for the indicated times and lysates examined by Western blot analysis for CDCP1, p-CDCP1-Y734, Src, p-Src-Y416 and GAPDH. Antibody dilution was 1:2,000 except the anti-GAPDH antibody which was 1:10,000. ( C ) Representative images of crystal violet stained colonies formed from HEY and OVMZ6-CDCP1 cells after treatment with the indicated concentrations of IgG, IgG-MMAE, 10D7 or 10D7-MMAE. ( D ) Graph of crystal violet staining, as a percentage of area (% Area), of colonies formed by HEY, OVMZ6-CDCP1 and OVMZ6 cells after treatment with increasing concentrations of IgG, IgG-MMAE, 10D7 or 10D7-MMAE. Data represent means ± SEM from three independent experiments. ***, P<0.001.

    Journal: Theranostics

    Article Title: Anti-CDCP1 immuno-conjugates for detection and inhibition of ovarian cancer

    doi: 10.7150/thno.30736

    Figure Lengend Snippet: 10D7-MMAE selectively inhibits colony formation of CDCP1 expressing but not non-expressing EOC cells. ( A ) Commassie stained gel of IgG and 10D7, and purified products from reactions of IgG and 10D7 with MMAE. ( B ) HEY cells were treated with 10D7-MMAE (5 µg/ml) for the indicated times and lysates examined by Western blot analysis for CDCP1, p-CDCP1-Y734, Src, p-Src-Y416 and GAPDH. Antibody dilution was 1:2,000 except the anti-GAPDH antibody which was 1:10,000. ( C ) Representative images of crystal violet stained colonies formed from HEY and OVMZ6-CDCP1 cells after treatment with the indicated concentrations of IgG, IgG-MMAE, 10D7 or 10D7-MMAE. ( D ) Graph of crystal violet staining, as a percentage of area (% Area), of colonies formed by HEY, OVMZ6-CDCP1 and OVMZ6 cells after treatment with increasing concentrations of IgG, IgG-MMAE, 10D7 or 10D7-MMAE. Data represent means ± SEM from three independent experiments. ***, P<0.001.

    Article Snippet: Rabbit anti-CDCP1 (#4115), rabbit anti-p-CDCP1-Y734 (#9050), mouse anti-Src (#2110) and rabbit anti-p-Src-Y416 family (#2101) antibodies were from Cell Signaling Technology (Gold Coast, Australia).

    Techniques: Expressing, Staining, Purification, Western Blot

    ( A ) Design of a PreScission Protease (Px)-cleavable CDCP1 ectodomain fused to a TEV-releasable Fc domain with C-terminal Avi-tag (CDCP1(Px)-Fc). The R368/K369 cleavage site was replaced with a Px recognition sequence (GS) 5 -LEVLFQGP-(GS) 5 . ( B ) SDS-PAGE of CDCP1 constructs. Px treatment cleaves CDCP1(Px)-Fc into NTF and CTF-Fc fragments. NTF is heavily glycosylated (predicted 14 N-linked glycosylation sites) and runs as a smeared higher-molecular weight band at ∼60 kDa. ( C ) SEC traces of CDCP1(R368/K369A)-Fc and CDCP1(Px)-Fc treated with Px, and NTF (TEV released) show that the NTF and CTF of CDCP1(Px) remain intact after proteolysis. Numbers denote fractions corresponding to the SDS-PAGE gel lanes in B . ( D ) BLI of IgG 4A06, which recognizes the NTF, shows robust binding to both Px-treated and untreated CDCP1(Px)-Fc. ( E ) Design of Px-cleavable CDCP1 with N-terminal FLAG-tag expressed on the surface of HEK293T cells. ( F ) Flow cytometry and western blot of HEK293T-wt, HEK293T-CDCP1(R368A/K369A), HEK293T-CDCP1(Px). Flow cytometry signal of anti-FLAG and IgG 4A06 remains unchanged with Px treatment. Western blot with anti-CDCP1 D1W9N, which recognizes the C-terminal intracellular region of CDCP1, confirms Px-mediated CDCP1 proteolysis at the intended molecular weight.

    Journal: bioRxiv

    Article Title: Targeting a proteolytic neo-epitope of CUB-domain containing protein 1 in RAS-driven cancer

    doi: 10.1101/2021.06.14.448427

    Figure Lengend Snippet: ( A ) Design of a PreScission Protease (Px)-cleavable CDCP1 ectodomain fused to a TEV-releasable Fc domain with C-terminal Avi-tag (CDCP1(Px)-Fc). The R368/K369 cleavage site was replaced with a Px recognition sequence (GS) 5 -LEVLFQGP-(GS) 5 . ( B ) SDS-PAGE of CDCP1 constructs. Px treatment cleaves CDCP1(Px)-Fc into NTF and CTF-Fc fragments. NTF is heavily glycosylated (predicted 14 N-linked glycosylation sites) and runs as a smeared higher-molecular weight band at ∼60 kDa. ( C ) SEC traces of CDCP1(R368/K369A)-Fc and CDCP1(Px)-Fc treated with Px, and NTF (TEV released) show that the NTF and CTF of CDCP1(Px) remain intact after proteolysis. Numbers denote fractions corresponding to the SDS-PAGE gel lanes in B . ( D ) BLI of IgG 4A06, which recognizes the NTF, shows robust binding to both Px-treated and untreated CDCP1(Px)-Fc. ( E ) Design of Px-cleavable CDCP1 with N-terminal FLAG-tag expressed on the surface of HEK293T cells. ( F ) Flow cytometry and western blot of HEK293T-wt, HEK293T-CDCP1(R368A/K369A), HEK293T-CDCP1(Px). Flow cytometry signal of anti-FLAG and IgG 4A06 remains unchanged with Px treatment. Western blot with anti-CDCP1 D1W9N, which recognizes the C-terminal intracellular region of CDCP1, confirms Px-mediated CDCP1 proteolysis at the intended molecular weight.

    Article Snippet: Immunoblotting was performed using CDCP1(D1W9N) (Cell Signaling, 13794S), Phospho-CDCP1(Tyr707) (Cell Signaling, 13111S), Phospho-CDCP1(Tyr806) (Cell Signaling, 13024S), Phospho-CDCP1(Try734) (Cell Signaling, 9050S), Phospho-CDCP1(Try743)(D2G2J) (Cell Signaling, 14965S), Src(36D10) (Cell Signaling, 2109S), Phospho Src Family (Tyr416) (Cell Signaling, 2101S), PKCδ (Cell Signaling, 2058S), Phospho-PKCdelta(Tyr311) (Cell Signaling, 2055S), alpha-Tubulin(DM1A) (Cell Signaling, 3873S), IRDye 680RD Goat anti-Mouse (LiCOR, 925–68070), and IRDye 800CW Goat anti-Rabbit (LiCOR, 926-32211) antibodies.

    Techniques: Sequencing, SDS Page, Construct, Molecular Weight, Binding Assay, FLAG-tag, Flow Cytometry, Western Blot

    ( A ) Schematic of IP-MS strategy to identify the endogenous proteolysis sites of CDCP1 on PDAC cells. CDCP1 was IP-ed with IgG 4A06 or D1W9N Ab and digested with Glu-C, which cleaves after aspartic acid, and was analyzed by LC-MS/MS to identify peptides corresponding to proteolytic products of CDCP1. ( B ) ( top ) Western blot of PDAC cell lines expressing differential amounts of uncleaved and cleaved CDCP1. D1W9N Ab was used to detect C-terminal fragment of CDCP1. PL5 and PL45 express mostly cleaved CDCP1, while HPAC expresses mostly uncleaved CDCP1. HPNE, a non-malignant pancreatic cell line, expresses low levels of CDCP1. ( bottom ) IP-blot shows that IP with IgG 4A06 can pull-down the CTF of CDCP1. ( C ) Depiction of peptides and proteolysis sites identified in PL5, PL45, and HPAC cell lines. Peptides identified by LC-MS for each cell line is aligned to the reference sequence highlighted in light blue. Aspartic acid residues recognized by Glu-C are highlighted in red underlined text. Three proteolysis sites of CDCP1: Cut 1 (K365), Cut 2 (R368), Cut 3 (K369), are observed in PL5 and PL45 cells but not on HPAC cells, and are highlighted in blue text and blue vertical lines.

    Journal: bioRxiv

    Article Title: Targeting a proteolytic neo-epitope of CUB-domain containing protein 1 in RAS-driven cancer

    doi: 10.1101/2021.06.14.448427

    Figure Lengend Snippet: ( A ) Schematic of IP-MS strategy to identify the endogenous proteolysis sites of CDCP1 on PDAC cells. CDCP1 was IP-ed with IgG 4A06 or D1W9N Ab and digested with Glu-C, which cleaves after aspartic acid, and was analyzed by LC-MS/MS to identify peptides corresponding to proteolytic products of CDCP1. ( B ) ( top ) Western blot of PDAC cell lines expressing differential amounts of uncleaved and cleaved CDCP1. D1W9N Ab was used to detect C-terminal fragment of CDCP1. PL5 and PL45 express mostly cleaved CDCP1, while HPAC expresses mostly uncleaved CDCP1. HPNE, a non-malignant pancreatic cell line, expresses low levels of CDCP1. ( bottom ) IP-blot shows that IP with IgG 4A06 can pull-down the CTF of CDCP1. ( C ) Depiction of peptides and proteolysis sites identified in PL5, PL45, and HPAC cell lines. Peptides identified by LC-MS for each cell line is aligned to the reference sequence highlighted in light blue. Aspartic acid residues recognized by Glu-C are highlighted in red underlined text. Three proteolysis sites of CDCP1: Cut 1 (K365), Cut 2 (R368), Cut 3 (K369), are observed in PL5 and PL45 cells but not on HPAC cells, and are highlighted in blue text and blue vertical lines.

    Article Snippet: Immunoblotting was performed using CDCP1(D1W9N) (Cell Signaling, 13794S), Phospho-CDCP1(Tyr707) (Cell Signaling, 13111S), Phospho-CDCP1(Tyr806) (Cell Signaling, 13024S), Phospho-CDCP1(Try734) (Cell Signaling, 9050S), Phospho-CDCP1(Try743)(D2G2J) (Cell Signaling, 14965S), Src(36D10) (Cell Signaling, 2109S), Phospho Src Family (Tyr416) (Cell Signaling, 2101S), PKCδ (Cell Signaling, 2058S), Phospho-PKCdelta(Tyr311) (Cell Signaling, 2055S), alpha-Tubulin(DM1A) (Cell Signaling, 3873S), IRDye 680RD Goat anti-Mouse (LiCOR, 925–68070), and IRDye 800CW Goat anti-Rabbit (LiCOR, 926-32211) antibodies.

    Techniques: Liquid Chromatography with Mass Spectroscopy, Western Blot, Expressing, Sequencing

    ( A ) Schematic of the co-transfection strategy to generate c-CDCP1 ectodomain. The NTF and CTF are encoded on separate plasmids with an IL2 secretion sequence. ( B ) SDS-PAGE of fl-CDCP1 and c-CDCP1 (Cut 1, Cut 2, Cut 3) ectodomain show successful expression and purification. NTF is heavily glycosylated and runs as a high-molecular weight smear. ( C ) BLI of IgG 4A06 to fl- or c-CDCP1 ectodomains show that the NTF of CDCP1 is intact on both cleaved and uncleaved CDCP1. ( D ) Differential Scanning Fluorimetry (DSF) shows that fl- and c-CDCP1 have similar melting profiles and stabilities, suggesting the NTF/CTF complex does not dissociate until full unfolding of the protein. T m is reported as an average and standard deviation of two replicates. ( E ) Circular Dichroism (CD) spectra of fl- and c-CDCP1. CDCP1 has a β-sheet signature with minima ∼217 nm. The slight difference in spectral shape between fl- and c-CDCP1 indicates a subtle change in secondary structure. ( F ) SAXS-derived P(r) function of fl- and c-CDCP1 ectodomains show similar overall architecture. SAXS-derived ab initio envelopes are shown under the graph. ( G ) Radii of gyration (R g ) of fl- and c-CDCP1. ( H ) SEC-MALS chromatograms of fl- and c-CDCP1 show similar elution profiles and molecular weights corresponding to monomeric ectodomain.

    Journal: bioRxiv

    Article Title: Targeting a proteolytic neo-epitope of CUB-domain containing protein 1 in RAS-driven cancer

    doi: 10.1101/2021.06.14.448427

    Figure Lengend Snippet: ( A ) Schematic of the co-transfection strategy to generate c-CDCP1 ectodomain. The NTF and CTF are encoded on separate plasmids with an IL2 secretion sequence. ( B ) SDS-PAGE of fl-CDCP1 and c-CDCP1 (Cut 1, Cut 2, Cut 3) ectodomain show successful expression and purification. NTF is heavily glycosylated and runs as a high-molecular weight smear. ( C ) BLI of IgG 4A06 to fl- or c-CDCP1 ectodomains show that the NTF of CDCP1 is intact on both cleaved and uncleaved CDCP1. ( D ) Differential Scanning Fluorimetry (DSF) shows that fl- and c-CDCP1 have similar melting profiles and stabilities, suggesting the NTF/CTF complex does not dissociate until full unfolding of the protein. T m is reported as an average and standard deviation of two replicates. ( E ) Circular Dichroism (CD) spectra of fl- and c-CDCP1. CDCP1 has a β-sheet signature with minima ∼217 nm. The slight difference in spectral shape between fl- and c-CDCP1 indicates a subtle change in secondary structure. ( F ) SAXS-derived P(r) function of fl- and c-CDCP1 ectodomains show similar overall architecture. SAXS-derived ab initio envelopes are shown under the graph. ( G ) Radii of gyration (R g ) of fl- and c-CDCP1. ( H ) SEC-MALS chromatograms of fl- and c-CDCP1 show similar elution profiles and molecular weights corresponding to monomeric ectodomain.

    Article Snippet: Immunoblotting was performed using CDCP1(D1W9N) (Cell Signaling, 13794S), Phospho-CDCP1(Tyr707) (Cell Signaling, 13111S), Phospho-CDCP1(Tyr806) (Cell Signaling, 13024S), Phospho-CDCP1(Try734) (Cell Signaling, 9050S), Phospho-CDCP1(Try743)(D2G2J) (Cell Signaling, 14965S), Src(36D10) (Cell Signaling, 2109S), Phospho Src Family (Tyr416) (Cell Signaling, 2101S), PKCδ (Cell Signaling, 2058S), Phospho-PKCdelta(Tyr311) (Cell Signaling, 2055S), alpha-Tubulin(DM1A) (Cell Signaling, 3873S), IRDye 680RD Goat anti-Mouse (LiCOR, 925–68070), and IRDye 800CW Goat anti-Rabbit (LiCOR, 926-32211) antibodies.

    Techniques: Cotransfection, Sequencing, SDS Page, Expressing, Purification, Molecular Weight, Standard Deviation, Derivative Assay

    ( A ) Schematic of strategy to generate HEK293T cell lines expressing fl- or c-CDCP1. For c-CDCP1, a lentiviral vector was designed where a T2A self-cleavage sequence flanks the CTF (res 370-836) and NTF (res 30-369). For fl-CDCP1, a lentiviral vector encoding the full CDCP1 sequence (res 30-836) was designed. An IL2 signal sequence precedes each fragment. ( B ) Flow cytometry of IgG 4A06 to HEK293T fl-CDCP1 and HEK293T c-CDCP1 cell lines indicates the NTF of CDCP1 is present on the cell surface for all cell lines. ( D ) Western blot of CDCP1 and intracellular proteins associated with CDCP1 signaling. Both fl-CDCP1 and c-CDCP1 are phosphorylated and initiate downstream signaling mediated by Src and PKCδ. Phosphorylation of Y734 on CDCP1 is important for phosphorylation of other tyrosine residues and downstream signaling partners. Note, anti-phosphoY311-PKCδ appears to be cross-reactive to CDCP1-pY734. ( D ) Cell adhesion assay comparing HEK239T fl-CDCP1 and c-CDCP1 shows that overexpression of both fl- and c- CDCP1 decreases cell adhesion and is dependent on phosphorylation of intracellular tyrosine residues, specifically of Y734. **p = 0.0016, ****p < 0.0001, ns = not significant p >0.05. (unpaired t-test)

    Journal: bioRxiv

    Article Title: Targeting a proteolytic neo-epitope of CUB-domain containing protein 1 in RAS-driven cancer

    doi: 10.1101/2021.06.14.448427

    Figure Lengend Snippet: ( A ) Schematic of strategy to generate HEK293T cell lines expressing fl- or c-CDCP1. For c-CDCP1, a lentiviral vector was designed where a T2A self-cleavage sequence flanks the CTF (res 370-836) and NTF (res 30-369). For fl-CDCP1, a lentiviral vector encoding the full CDCP1 sequence (res 30-836) was designed. An IL2 signal sequence precedes each fragment. ( B ) Flow cytometry of IgG 4A06 to HEK293T fl-CDCP1 and HEK293T c-CDCP1 cell lines indicates the NTF of CDCP1 is present on the cell surface for all cell lines. ( D ) Western blot of CDCP1 and intracellular proteins associated with CDCP1 signaling. Both fl-CDCP1 and c-CDCP1 are phosphorylated and initiate downstream signaling mediated by Src and PKCδ. Phosphorylation of Y734 on CDCP1 is important for phosphorylation of other tyrosine residues and downstream signaling partners. Note, anti-phosphoY311-PKCδ appears to be cross-reactive to CDCP1-pY734. ( D ) Cell adhesion assay comparing HEK239T fl-CDCP1 and c-CDCP1 shows that overexpression of both fl- and c- CDCP1 decreases cell adhesion and is dependent on phosphorylation of intracellular tyrosine residues, specifically of Y734. **p = 0.0016, ****p < 0.0001, ns = not significant p >0.05. (unpaired t-test)

    Article Snippet: Immunoblotting was performed using CDCP1(D1W9N) (Cell Signaling, 13794S), Phospho-CDCP1(Tyr707) (Cell Signaling, 13111S), Phospho-CDCP1(Tyr806) (Cell Signaling, 13024S), Phospho-CDCP1(Try734) (Cell Signaling, 9050S), Phospho-CDCP1(Try743)(D2G2J) (Cell Signaling, 14965S), Src(36D10) (Cell Signaling, 2109S), Phospho Src Family (Tyr416) (Cell Signaling, 2101S), PKCδ (Cell Signaling, 2058S), Phospho-PKCdelta(Tyr311) (Cell Signaling, 2055S), alpha-Tubulin(DM1A) (Cell Signaling, 3873S), IRDye 680RD Goat anti-Mouse (LiCOR, 925–68070), and IRDye 800CW Goat anti-Rabbit (LiCOR, 926-32211) antibodies.

    Techniques: Expressing, Plasmid Preparation, Sequencing, Flow Cytometry, Western Blot, Cell Adhesion Assay, Over Expression

    ( A ) Differential phage selection strategy to identify a cleaved CDCP1-specific antibody. Fab-phage were pre-cleared with fl-CDCP1-Fc prior to positive selection with c-CDCP1-Fc. Enriched Fab-phage were characterized by phage ELISA for selective binding to c-CDCP1-Fc. ( B ) BLI show specific binding of IgG CL03 to c-CDCP1-Fc but not to fl-CDCP1-Fc. (K D = 150-840 pM, Table S1 ) ( C ) Negative-stain EM 3D reconstruction of c-CDCP1 with CL03 Fab. ( left ) 2D class averages of c-CDCP1(Cut3) + CL03 Fab in the absence and presence of anti-Fab V H H. ( right ) Different views of 3D EM map of CDCP1(Cut3) + CL03 Fab + V H H with crystal structure of Fab (green) and V H H (blue) modeled into the density. ( D ) Immunofluorescence of Alexa Fluor-488-labeled IgG CL03 ( left panels ) and IgG 4A06 ( right panels ) on HPAC, PL5, and HPNE cells. IgG CL03 specifically stains PL5 cells that express cleaved CDCP1, while IgG 4A06 stains both HPAC and PL5 cells. ( E ) Flow cytometry shows that IgG CL03 binds to cleaved CDCP1-expressing PL5 and PL45 cells but not HPAC or HPNE cells. (n = 3, data represent average and standard deviation). ( F ) ( top ) Schematic of antibody drug conjugate (ADC) cell killing assay. ( bottom ) Dose-dependent ADC-mediated cell killing with IgG CL03 was only observed against PL5 and PL45 cells that express cleaved CDCP1, and only in the presence of both the primary and secondary antibody. (**p = 0.004, ***p = 0.0018, unpaired T-test) ( G ) In vivo PET imaging of 89 Zr-labeled IgG CL03 in PDAC xenograft mice harboring PL5 tumors (n = 4).

    Journal: bioRxiv

    Article Title: Targeting a proteolytic neo-epitope of CUB-domain containing protein 1 in RAS-driven cancer

    doi: 10.1101/2021.06.14.448427

    Figure Lengend Snippet: ( A ) Differential phage selection strategy to identify a cleaved CDCP1-specific antibody. Fab-phage were pre-cleared with fl-CDCP1-Fc prior to positive selection with c-CDCP1-Fc. Enriched Fab-phage were characterized by phage ELISA for selective binding to c-CDCP1-Fc. ( B ) BLI show specific binding of IgG CL03 to c-CDCP1-Fc but not to fl-CDCP1-Fc. (K D = 150-840 pM, Table S1 ) ( C ) Negative-stain EM 3D reconstruction of c-CDCP1 with CL03 Fab. ( left ) 2D class averages of c-CDCP1(Cut3) + CL03 Fab in the absence and presence of anti-Fab V H H. ( right ) Different views of 3D EM map of CDCP1(Cut3) + CL03 Fab + V H H with crystal structure of Fab (green) and V H H (blue) modeled into the density. ( D ) Immunofluorescence of Alexa Fluor-488-labeled IgG CL03 ( left panels ) and IgG 4A06 ( right panels ) on HPAC, PL5, and HPNE cells. IgG CL03 specifically stains PL5 cells that express cleaved CDCP1, while IgG 4A06 stains both HPAC and PL5 cells. ( E ) Flow cytometry shows that IgG CL03 binds to cleaved CDCP1-expressing PL5 and PL45 cells but not HPAC or HPNE cells. (n = 3, data represent average and standard deviation). ( F ) ( top ) Schematic of antibody drug conjugate (ADC) cell killing assay. ( bottom ) Dose-dependent ADC-mediated cell killing with IgG CL03 was only observed against PL5 and PL45 cells that express cleaved CDCP1, and only in the presence of both the primary and secondary antibody. (**p = 0.004, ***p = 0.0018, unpaired T-test) ( G ) In vivo PET imaging of 89 Zr-labeled IgG CL03 in PDAC xenograft mice harboring PL5 tumors (n = 4).

    Article Snippet: Immunoblotting was performed using CDCP1(D1W9N) (Cell Signaling, 13794S), Phospho-CDCP1(Tyr707) (Cell Signaling, 13111S), Phospho-CDCP1(Tyr806) (Cell Signaling, 13024S), Phospho-CDCP1(Try734) (Cell Signaling, 9050S), Phospho-CDCP1(Try743)(D2G2J) (Cell Signaling, 14965S), Src(36D10) (Cell Signaling, 2109S), Phospho Src Family (Tyr416) (Cell Signaling, 2101S), PKCδ (Cell Signaling, 2058S), Phospho-PKCdelta(Tyr311) (Cell Signaling, 2055S), alpha-Tubulin(DM1A) (Cell Signaling, 3873S), IRDye 680RD Goat anti-Mouse (LiCOR, 925–68070), and IRDye 800CW Goat anti-Rabbit (LiCOR, 926-32211) antibodies.

    Techniques: Selection, Enzyme-linked Immunosorbent Assay, Binding Assay, Staining, Immunofluorescence, Labeling, Flow Cytometry, Expressing, Standard Deviation, In Vivo, Imaging

    ( A ) BLI show specific binding of IgG58 to mouse c-CDCP1-Fc, but not to fl-CDCP1-Fc. ( B ) Flow cytometry shows that IgG58 binds robustly to Fc1245 c-CDCP1, but not to Fc1245 WT cells (n = 3, error bars represent s.d.). ( C ) Dose-dependent ADC-mediated cell killing with IgG58-MMAF treatment was only observed with Fc1245 c-CDCP1 cells and not Fc1245 WT cells. (n = 2, error bars represent s.d.) (**p = 0.002, unpaired T-test) ( D ) Representative in vivo PET images of 89 Zr-IgG58 and 89 Zr-IgG12 in mice harboring subcutaneous Fc1245 c-CDCP1 tumors. Co-administration of 50X unlabeled IgG was used to examine target specificity. ( E ) Biodistribution of 89 Zr-IgG58 and 89 Zr-IgG12 in mice harboring subcutaneous Fc1245 c-CDCP1 tumors (n = 5 per arm). Both 89 Zr-IgG58 and 89 Zr-IgG12 signal decreased when 50X unlabeled IgG was administered, indicating target-specific localization (**p = 0.003, ***p = 0.0002, unpaired T-test). 89 Zr-IgG58 shows stronger signal in the tumor, while 89 Zr-IgG12 shows weaker tumor localization and more widespread normal tissue distribution (****p <0.0001, unpaired T-test). ( F ) ADC toxicity assay in non-tumor bearing mice. Mice (n = 5 per arm) were dosed weekly with 5, 10, 15 mg/kg of either IgG12-MMAF or IgG58-MMAF and body weight was monitored for treatment-associated toxicity. There was a significant difference between the treatment arms (F(5,32) = 3.11, p = 0.0002, ANOVA), with IgG58-MMAF treatment being better tolerated, with significant differences between IgG12-MMAF and IgG58-MMAF treatments at the 15 mg/kg dose (***p = 0.0068) and 10 mg/kg dose (**p = 0.0067) (Tukey’s multiple comparisons test). ( G-H ) Theranostic efficacy study of 177 Lu-IgG58. Mice (n = 5 per treatment arm, n = 8 for vehicle arm) were injected with 400 µCi of 177 Lu-IgG58 or vehicle 4 days after Fc1245-c-CDCP1 tumor implantation. For the 2-dose arm, the treatment was repeated 6 days later. Treatment with 177 Lu-IgG58 resulted in decreased tumor growth and increased survival compared to the vehicle arm (***p = 0.0008, ****p <0.0001, unpaired two-tailed T-test).

    Journal: bioRxiv

    Article Title: Targeting a proteolytic neo-epitope of CUB-domain containing protein 1 in RAS-driven cancer

    doi: 10.1101/2021.06.14.448427

    Figure Lengend Snippet: ( A ) BLI show specific binding of IgG58 to mouse c-CDCP1-Fc, but not to fl-CDCP1-Fc. ( B ) Flow cytometry shows that IgG58 binds robustly to Fc1245 c-CDCP1, but not to Fc1245 WT cells (n = 3, error bars represent s.d.). ( C ) Dose-dependent ADC-mediated cell killing with IgG58-MMAF treatment was only observed with Fc1245 c-CDCP1 cells and not Fc1245 WT cells. (n = 2, error bars represent s.d.) (**p = 0.002, unpaired T-test) ( D ) Representative in vivo PET images of 89 Zr-IgG58 and 89 Zr-IgG12 in mice harboring subcutaneous Fc1245 c-CDCP1 tumors. Co-administration of 50X unlabeled IgG was used to examine target specificity. ( E ) Biodistribution of 89 Zr-IgG58 and 89 Zr-IgG12 in mice harboring subcutaneous Fc1245 c-CDCP1 tumors (n = 5 per arm). Both 89 Zr-IgG58 and 89 Zr-IgG12 signal decreased when 50X unlabeled IgG was administered, indicating target-specific localization (**p = 0.003, ***p = 0.0002, unpaired T-test). 89 Zr-IgG58 shows stronger signal in the tumor, while 89 Zr-IgG12 shows weaker tumor localization and more widespread normal tissue distribution (****p <0.0001, unpaired T-test). ( F ) ADC toxicity assay in non-tumor bearing mice. Mice (n = 5 per arm) were dosed weekly with 5, 10, 15 mg/kg of either IgG12-MMAF or IgG58-MMAF and body weight was monitored for treatment-associated toxicity. There was a significant difference between the treatment arms (F(5,32) = 3.11, p = 0.0002, ANOVA), with IgG58-MMAF treatment being better tolerated, with significant differences between IgG12-MMAF and IgG58-MMAF treatments at the 15 mg/kg dose (***p = 0.0068) and 10 mg/kg dose (**p = 0.0067) (Tukey’s multiple comparisons test). ( G-H ) Theranostic efficacy study of 177 Lu-IgG58. Mice (n = 5 per treatment arm, n = 8 for vehicle arm) were injected with 400 µCi of 177 Lu-IgG58 or vehicle 4 days after Fc1245-c-CDCP1 tumor implantation. For the 2-dose arm, the treatment was repeated 6 days later. Treatment with 177 Lu-IgG58 resulted in decreased tumor growth and increased survival compared to the vehicle arm (***p = 0.0008, ****p <0.0001, unpaired two-tailed T-test).

    Article Snippet: Immunoblotting was performed using CDCP1(D1W9N) (Cell Signaling, 13794S), Phospho-CDCP1(Tyr707) (Cell Signaling, 13111S), Phospho-CDCP1(Tyr806) (Cell Signaling, 13024S), Phospho-CDCP1(Try734) (Cell Signaling, 9050S), Phospho-CDCP1(Try743)(D2G2J) (Cell Signaling, 14965S), Src(36D10) (Cell Signaling, 2109S), Phospho Src Family (Tyr416) (Cell Signaling, 2101S), PKCδ (Cell Signaling, 2058S), Phospho-PKCdelta(Tyr311) (Cell Signaling, 2055S), alpha-Tubulin(DM1A) (Cell Signaling, 3873S), IRDye 680RD Goat anti-Mouse (LiCOR, 925–68070), and IRDye 800CW Goat anti-Rabbit (LiCOR, 926-32211) antibodies.

    Techniques: Binding Assay, Flow Cytometry, In Vivo, Injection, Tumor Implantation, Two Tailed Test

    (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

    ( 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.

    Journal: bioRxiv

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

    doi: 10.1101/789339

    Figure Lengend 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.

    Article Snippet: For this study, antibodies against CDCP1 (4115), CDCP1 pY734 (9050), STAT3 (9132), STAT3 pY705 (9145), Myc-tag (2276), Met pY1234/1235 (3077), and Met (8198) were purchased from Cell Signaling Technologies.

    Techniques: 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.

    Journal: bioRxiv

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

    doi: 10.1101/789339

    Figure Lengend 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.

    Article Snippet: For this study, antibodies against CDCP1 (4115), CDCP1 pY734 (9050), STAT3 (9132), STAT3 pY705 (9145), Myc-tag (2276), Met pY1234/1235 (3077), and Met (8198) were purchased from Cell Signaling Technologies.

    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. 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).

    Journal: bioRxiv

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

    doi: 10.1101/789339

    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. 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).

    Article Snippet: For this study, antibodies against CDCP1 (4115), CDCP1 pY734 (9050), STAT3 (9132), STAT3 pY705 (9145), Myc-tag (2276), Met pY1234/1235 (3077), and Met (8198) were purchased from Cell Signaling Technologies.

    Techniques: 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.

    Journal: bioRxiv

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

    doi: 10.1101/789339

    Figure Lengend 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.

    Article Snippet: For this study, antibodies against CDCP1 (4115), CDCP1 pY734 (9050), STAT3 (9132), STAT3 pY705 (9145), Myc-tag (2276), Met pY1234/1235 (3077), and Met (8198) were purchased from Cell Signaling Technologies.

    Techniques: Cell Culture, Incubation, Staining

    Journal: bioRxiv

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

    doi: 10.1101/789339

    Figure Lengend Snippet:

    Article Snippet: For this study, antibodies against CDCP1 (4115), CDCP1 pY734 (9050), STAT3 (9132), STAT3 pY705 (9145), Myc-tag (2276), Met pY1234/1235 (3077), and Met (8198) were purchased from Cell Signaling Technologies.

    Techniques:

    ( 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.

    Journal: bioRxiv

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

    doi: 10.1101/789339

    Figure Lengend 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.

    Article Snippet: For this study, antibodies against CDCP1 (4115), CDCP1 pY734 (9050), STAT3 (9132), STAT3 pY705 (9145), Myc-tag (2276), Met pY1234/1235 (3077), and Met (8198) were purchased from Cell Signaling Technologies.

    Techniques: 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.

    Journal: bioRxiv

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

    doi: 10.1101/789339

    Figure Lengend 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.

    Article Snippet: For this study, antibodies against CDCP1 (4115), CDCP1 pY734 (9050), STAT3 (9132), STAT3 pY705 (9145), Myc-tag (2276), Met pY1234/1235 (3077), and Met (8198) were purchased from Cell Signaling Technologies.

    Techniques: 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.

    Journal: bioRxiv

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

    doi: 10.1101/789339

    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 ) 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.

    Article Snippet: For this study, antibodies against CDCP1 (4115), CDCP1 pY734 (9050), STAT3 (9132), STAT3 pY705 (9145), Myc-tag (2276), Met pY1234/1235 (3077), and Met (8198) were purchased from Cell Signaling Technologies.

    Techniques: 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.

    Journal: bioRxiv

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

    doi: 10.1101/789339

    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 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.

    Article Snippet: For this study, antibodies against CDCP1 (4115), CDCP1 pY734 (9050), STAT3 (9132), STAT3 pY705 (9145), Myc-tag (2276), Met pY1234/1235 (3077), and Met (8198) were purchased from Cell Signaling Technologies.

    Techniques: 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.

    Journal: bioRxiv

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

    doi: 10.1101/789339

    Figure Lengend 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.

    Article Snippet: For this study, antibodies against CDCP1 (4115), CDCP1 pY734 (9050), STAT3 (9132), STAT3 pY705 (9145), Myc-tag (2276), Met pY1234/1235 (3077), and Met (8198) were purchased from Cell Signaling Technologies.

    Techniques: 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.

    Journal: bioRxiv

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

    doi: 10.1101/789339

    Figure Lengend 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.

    Article Snippet: For this study, antibodies against CDCP1 (4115), CDCP1 pY734 (9050), STAT3 (9132), STAT3 pY705 (9145), Myc-tag (2276), Met pY1234/1235 (3077), and Met (8198) were purchased from Cell Signaling Technologies.

    Techniques: 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

    Journal: bioRxiv

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

    doi: 10.1101/789339

    Figure Lengend 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

    Article Snippet: For this study, antibodies against CDCP1 (4115), CDCP1 pY734 (9050), STAT3 (9132), STAT3 pY705 (9145), Myc-tag (2276), Met pY1234/1235 (3077), and Met (8198) were purchased from Cell Signaling Technologies.

    Techniques: 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.

    Journal: bioRxiv

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

    doi: 10.1101/789339

    Figure Lengend 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.

    Article Snippet: For this study, antibodies against CDCP1 (4115), CDCP1 pY734 (9050), STAT3 (9132), STAT3 pY705 (9145), Myc-tag (2276), Met pY1234/1235 (3077), and Met (8198) were purchased from Cell Signaling Technologies.

    Techniques: 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.

    Journal: bioRxiv

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

    doi: 10.1101/789339

    Figure Lengend 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.

    Article Snippet: For this study, antibodies against CDCP1 (4115), CDCP1 pY734 (9050), STAT3 (9132), STAT3 pY705 (9145), Myc-tag (2276), Met pY1234/1235 (3077), and Met (8198) were purchased from Cell Signaling Technologies.

    Techniques: 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.

    Journal: bioRxiv

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

    doi: 10.1101/789339

    Figure Lengend 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.

    Article Snippet: For this study, antibodies against CDCP1 (4115), CDCP1 pY734 (9050), STAT3 (9132), STAT3 pY705 (9145), Myc-tag (2276), Met pY1234/1235 (3077), and Met (8198) were purchased from Cell Signaling Technologies.

    Techniques: 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.

    Journal: bioRxiv

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

    doi: 10.1101/789339

    Figure Lengend 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.

    Article Snippet: For this study, antibodies against CDCP1 (4115), CDCP1 pY734 (9050), STAT3 (9132), STAT3 pY705 (9145), Myc-tag (2276), Met pY1234/1235 (3077), and Met (8198) were purchased from Cell Signaling Technologies.

    Techniques: 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.

    Journal: bioRxiv

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

    doi: 10.1101/789339

    Figure Lengend 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.

    Article Snippet: For this study, antibodies against CDCP1 (4115), CDCP1 pY734 (9050), STAT3 (9132), STAT3 pY705 (9145), Myc-tag (2276), Met pY1234/1235 (3077), and Met (8198) were purchased from Cell Signaling Technologies.

    Techniques: Expressing

    PDGF-BB stimulation upregulates CDCP1 in TNBC cells. a CDCP1 in MDA-MB-231 cells treated with various growth factors, cytokines, and chemokines for 48 h was analyzed by FACS and reported as percentage upregulation with respect to the maximum increase induced by WHFs (100%). Representative experiment. b WB analysis of CDCP1, phospho-CDCP1 (Y734), PDGFRβ, and phospho-PDGFRβ (Y751) in MDA-MB-231 cells treated with or without PDGF-BB 20 ng/ml for 48 h. The fold-change increase in phospho-CDCP1 and CDCP1, calculated by densitometry, was 1.6 and 1.9, respectively. c WB analysis of CDCP1 in MDA-MB-231 cells treated with or without PDGF-BB 20 ng/ml and/or cycloheximide (1 μM) for 24 h. Monoclonal anti-actin was used as the total protein loading control

    Journal: BMC Cancer

    Article Title: The PDGFRβ/ERK1/2 pathway regulates CDCP1 expression in triple-negative breast cancer

    doi: 10.1186/s12885-018-4500-9

    Figure Lengend Snippet: PDGF-BB stimulation upregulates CDCP1 in TNBC cells. a CDCP1 in MDA-MB-231 cells treated with various growth factors, cytokines, and chemokines for 48 h was analyzed by FACS and reported as percentage upregulation with respect to the maximum increase induced by WHFs (100%). Representative experiment. b WB analysis of CDCP1, phospho-CDCP1 (Y734), PDGFRβ, and phospho-PDGFRβ (Y751) in MDA-MB-231 cells treated with or without PDGF-BB 20 ng/ml for 48 h. The fold-change increase in phospho-CDCP1 and CDCP1, calculated by densitometry, was 1.6 and 1.9, respectively. c WB analysis of CDCP1 in MDA-MB-231 cells treated with or without PDGF-BB 20 ng/ml and/or cycloheximide (1 μM) for 24 h. Monoclonal anti-actin was used as the total protein loading control

    Article Snippet: Biochemical analyses were performed using rabbit polyclonal antibodies against CDCP1, phospho-CDCP1 (Tyr734), p44/42 MAPK (ERK1/2), phospho-p44/42 MAPK (ERK1/2) (Thr202/Tyr204) (Cell Signaling, Danvers, MA), and PDGFRβ (Santa Cruz Biotechnology, Dallas, TX) or mouse monoclonal anti-phospho-PDGFRβ (Tyr751) (clone 88H8) (Cell Signaling); polyclonal anti-rabbit or -mouse IgG (GE Healthcare, Chicago, IL) was the secondary antibody.

    Techniques:

    PDGFRβ regulates CDCP1 expression in TNBC cells. a WB analysis of PDGFRβ and CDCP1 in MDA-MB-231 cells transfected with 100 nM PDGFRβ siRNA or the appropriate negative control. Cells were harvested at 48 h post-transfection. b WB analysis of CDCP1 and phosphoERK1/2 (T202/Y204) in MDA-MB-231 cells transfected with 100 nM PDGFRβ siRNA or the appropriate negative control and with or without 5% WHF in culture medium for 24 h. Dottes lines demarcate juxtaposed images originating from separate lines of the same western blot. The fold-change increase in CDCP1, calculated by densitometry, was 2.3 and 1.6, respectively. Monoclonal anti-actin was used as the total protein loading control

    Journal: BMC Cancer

    Article Title: The PDGFRβ/ERK1/2 pathway regulates CDCP1 expression in triple-negative breast cancer

    doi: 10.1186/s12885-018-4500-9

    Figure Lengend Snippet: PDGFRβ regulates CDCP1 expression in TNBC cells. a WB analysis of PDGFRβ and CDCP1 in MDA-MB-231 cells transfected with 100 nM PDGFRβ siRNA or the appropriate negative control. Cells were harvested at 48 h post-transfection. b WB analysis of CDCP1 and phosphoERK1/2 (T202/Y204) in MDA-MB-231 cells transfected with 100 nM PDGFRβ siRNA or the appropriate negative control and with or without 5% WHF in culture medium for 24 h. Dottes lines demarcate juxtaposed images originating from separate lines of the same western blot. The fold-change increase in CDCP1, calculated by densitometry, was 2.3 and 1.6, respectively. Monoclonal anti-actin was used as the total protein loading control

    Article Snippet: Biochemical analyses were performed using rabbit polyclonal antibodies against CDCP1, phospho-CDCP1 (Tyr734), p44/42 MAPK (ERK1/2), phospho-p44/42 MAPK (ERK1/2) (Thr202/Tyr204) (Cell Signaling, Danvers, MA), and PDGFRβ (Santa Cruz Biotechnology, Dallas, TX) or mouse monoclonal anti-phospho-PDGFRβ (Tyr751) (clone 88H8) (Cell Signaling); polyclonal anti-rabbit or -mouse IgG (GE Healthcare, Chicago, IL) was the secondary antibody.

    Techniques: Expressing, Transfection, Negative Control, Western Blot

    ERK1/2 activity regulates CDCP1 expression in TNBC cells. a WB analysis of phospho-ERK1/2 (T202/Y204) in MDA-MB-231 cells treated with or without 20 ng/ml PDGF-BB for 10 and 60 min. b WB analysis of CDCP1 and phosphoERK1/2 (T202/Y204) in MDA-MB-231 cells treated with or without the ERK1/2 inhibitor UO126 (2 μM) and stimulated with or without 20 ng/ml PDGF-BB for 24 h. c WB analysis of CDCP1 in MDA-MB 231 cells treated with or without UO126 (2 μM) and stimulated with or without 5% WHF in culture medium for 24 h. Dotted lines demarcate juxtaposed images originating from separate lines of the same western blot. d WB analysis of CDCP1, phosphoERK1/2 (T202/Y204), and ERK1/2 in SUM149, SUM159, MDA-MB468, BT-549, MDA-MB-231, and HCC1937 cells treated with or without UO126 (2 μM) under standard medium conditions for 24 h. Monoclonal anti-actin was used as the total protein loading control

    Journal: BMC Cancer

    Article Title: The PDGFRβ/ERK1/2 pathway regulates CDCP1 expression in triple-negative breast cancer

    doi: 10.1186/s12885-018-4500-9

    Figure Lengend Snippet: ERK1/2 activity regulates CDCP1 expression in TNBC cells. a WB analysis of phospho-ERK1/2 (T202/Y204) in MDA-MB-231 cells treated with or without 20 ng/ml PDGF-BB for 10 and 60 min. b WB analysis of CDCP1 and phosphoERK1/2 (T202/Y204) in MDA-MB-231 cells treated with or without the ERK1/2 inhibitor UO126 (2 μM) and stimulated with or without 20 ng/ml PDGF-BB for 24 h. c WB analysis of CDCP1 in MDA-MB 231 cells treated with or without UO126 (2 μM) and stimulated with or without 5% WHF in culture medium for 24 h. Dotted lines demarcate juxtaposed images originating from separate lines of the same western blot. d WB analysis of CDCP1, phosphoERK1/2 (T202/Y204), and ERK1/2 in SUM149, SUM159, MDA-MB468, BT-549, MDA-MB-231, and HCC1937 cells treated with or without UO126 (2 μM) under standard medium conditions for 24 h. Monoclonal anti-actin was used as the total protein loading control

    Article Snippet: Biochemical analyses were performed using rabbit polyclonal antibodies against CDCP1, phospho-CDCP1 (Tyr734), p44/42 MAPK (ERK1/2), phospho-p44/42 MAPK (ERK1/2) (Thr202/Tyr204) (Cell Signaling, Danvers, MA), and PDGFRβ (Santa Cruz Biotechnology, Dallas, TX) or mouse monoclonal anti-phospho-PDGFRβ (Tyr751) (clone 88H8) (Cell Signaling); polyclonal anti-rabbit or -mouse IgG (GE Healthcare, Chicago, IL) was the secondary antibody.

    Techniques: Activity Assay, Expressing, Western Blot

    Schematic representation of CDCP1 upregulation induced by PDGF-BB/PDGFRβ pathway through ERK1/2 activation. PDGFRβ dimerizes and is activated upon binding of the PDGF-BB ligand, causing the activation of the kinase domain, visualized as tyrosine phosphorylation (P) of the receptor molecules. In conjunction with dimerization and kinase activation, the receptor molecules undergoes a conformational changes, which allow a basal kinase activity, leading to full enzymatic activity directed toward downstream mediators such as ERK1/2. ERK1/2 activity is necessary for CDCP1 protein neo-synthesis, as demonstrated by the reduction of CDCP1 protein levels in presence of UO126, an inhibitor of ERK1/2

    Journal: BMC Cancer

    Article Title: The PDGFRβ/ERK1/2 pathway regulates CDCP1 expression in triple-negative breast cancer

    doi: 10.1186/s12885-018-4500-9

    Figure Lengend Snippet: Schematic representation of CDCP1 upregulation induced by PDGF-BB/PDGFRβ pathway through ERK1/2 activation. PDGFRβ dimerizes and is activated upon binding of the PDGF-BB ligand, causing the activation of the kinase domain, visualized as tyrosine phosphorylation (P) of the receptor molecules. In conjunction with dimerization and kinase activation, the receptor molecules undergoes a conformational changes, which allow a basal kinase activity, leading to full enzymatic activity directed toward downstream mediators such as ERK1/2. ERK1/2 activity is necessary for CDCP1 protein neo-synthesis, as demonstrated by the reduction of CDCP1 protein levels in presence of UO126, an inhibitor of ERK1/2

    Article Snippet: Biochemical analyses were performed using rabbit polyclonal antibodies against CDCP1, phospho-CDCP1 (Tyr734), p44/42 MAPK (ERK1/2), phospho-p44/42 MAPK (ERK1/2) (Thr202/Tyr204) (Cell Signaling, Danvers, MA), and PDGFRβ (Santa Cruz Biotechnology, Dallas, TX) or mouse monoclonal anti-phospho-PDGFRβ (Tyr751) (clone 88H8) (Cell Signaling); polyclonal anti-rabbit or -mouse IgG (GE Healthcare, Chicago, IL) was the secondary antibody.

    Techniques: Activation Assay, Binding Assay, Activity Assay

    Clinical characteristics of TNBC patients according to expression of PDGFRβ and  CDCP1

    Journal: BMC Cancer

    Article Title: The PDGFRβ/ERK1/2 pathway regulates CDCP1 expression in triple-negative breast cancer

    doi: 10.1186/s12885-018-4500-9

    Figure Lengend Snippet: Clinical characteristics of TNBC patients according to expression of PDGFRβ and CDCP1

    Article Snippet: Biochemical analyses were performed using rabbit polyclonal antibodies against CDCP1, phospho-CDCP1 (Tyr734), p44/42 MAPK (ERK1/2), phospho-p44/42 MAPK (ERK1/2) (Thr202/Tyr204) (Cell Signaling, Danvers, MA), and PDGFRβ (Santa Cruz Biotechnology, Dallas, TX) or mouse monoclonal anti-phospho-PDGFRβ (Tyr751) (clone 88H8) (Cell Signaling); polyclonal anti-rabbit or -mouse IgG (GE Healthcare, Chicago, IL) was the secondary antibody.

    Techniques: Expressing

    IHC staining of PDGFRβ and CDCP1 in TNBC. FFPE sections of TNBC specimens were analyzed by IHC for PDGFRβ and CDCP1. a Representative image of a PDGFRβ- and CDCP1-positive case, with plasma membrane staining, at 10X and 40X magnification; b Representative image of a PDGFRβ- and CDCP1-negative case, at 10X and 40X magnification

    Journal: BMC Cancer

    Article Title: The PDGFRβ/ERK1/2 pathway regulates CDCP1 expression in triple-negative breast cancer

    doi: 10.1186/s12885-018-4500-9

    Figure Lengend Snippet: IHC staining of PDGFRβ and CDCP1 in TNBC. FFPE sections of TNBC specimens were analyzed by IHC for PDGFRβ and CDCP1. a Representative image of a PDGFRβ- and CDCP1-positive case, with plasma membrane staining, at 10X and 40X magnification; b Representative image of a PDGFRβ- and CDCP1-negative case, at 10X and 40X magnification

    Article Snippet: Biochemical analyses were performed using rabbit polyclonal antibodies against CDCP1, phospho-CDCP1 (Tyr734), p44/42 MAPK (ERK1/2), phospho-p44/42 MAPK (ERK1/2) (Thr202/Tyr204) (Cell Signaling, Danvers, MA), and PDGFRβ (Santa Cruz Biotechnology, Dallas, TX) or mouse monoclonal anti-phospho-PDGFRβ (Tyr751) (clone 88H8) (Cell Signaling); polyclonal anti-rabbit or -mouse IgG (GE Healthcare, Chicago, IL) was the secondary antibody.

    Techniques: Immunohistochemistry, Staining

    PDGFRβ expression in tumor and vascular lacunae according to expression of CDCP1 (IHC CDCP1)

    Journal: BMC Cancer

    Article Title: The PDGFRβ/ERK1/2 pathway regulates CDCP1 expression in triple-negative breast cancer

    doi: 10.1186/s12885-018-4500-9

    Figure Lengend Snippet: PDGFRβ expression in tumor and vascular lacunae according to expression of CDCP1 (IHC CDCP1)

    Article Snippet: Biochemical analyses were performed using rabbit polyclonal antibodies against CDCP1, phospho-CDCP1 (Tyr734), p44/42 MAPK (ERK1/2), phospho-p44/42 MAPK (ERK1/2) (Thr202/Tyr204) (Cell Signaling, Danvers, MA), and PDGFRβ (Santa Cruz Biotechnology, Dallas, TX) or mouse monoclonal anti-phospho-PDGFRβ (Tyr751) (clone 88H8) (Cell Signaling); polyclonal anti-rabbit or -mouse IgG (GE Healthcare, Chicago, IL) was the secondary antibody.

    Techniques: Expressing

    Genetic alterations of CDCP1 in TNBC. FFPE sections of TNBC specimens were analyzed by dual-color FISH using for CDCP1 genetic alteration CDCP1/CEP3 probes on a FFPE sections of TNBC specimens FISH. a Representative image of TNBC specimen positive for CDCP1 IHC staining, showing tumor cells with > 3 red signals for CEP3 and > 3 green signals for the CDCP1 locus (polysomy); b Representative image of TNBC specimen negative for CDCP1 IHC staining, showing tumor cells with > 3 signals for CEP3 and < 3 green signals for the CDCP1 locus (deletion)

    Journal: BMC Cancer

    Article Title: The PDGFRβ/ERK1/2 pathway regulates CDCP1 expression in triple-negative breast cancer

    doi: 10.1186/s12885-018-4500-9

    Figure Lengend Snippet: Genetic alterations of CDCP1 in TNBC. FFPE sections of TNBC specimens were analyzed by dual-color FISH using for CDCP1 genetic alteration CDCP1/CEP3 probes on a FFPE sections of TNBC specimens FISH. a Representative image of TNBC specimen positive for CDCP1 IHC staining, showing tumor cells with > 3 red signals for CEP3 and > 3 green signals for the CDCP1 locus (polysomy); b Representative image of TNBC specimen negative for CDCP1 IHC staining, showing tumor cells with > 3 signals for CEP3 and < 3 green signals for the CDCP1 locus (deletion)

    Article Snippet: Biochemical analyses were performed using rabbit polyclonal antibodies against CDCP1, phospho-CDCP1 (Tyr734), p44/42 MAPK (ERK1/2), phospho-p44/42 MAPK (ERK1/2) (Thr202/Tyr204) (Cell Signaling, Danvers, MA), and PDGFRβ (Santa Cruz Biotechnology, Dallas, TX) or mouse monoclonal anti-phospho-PDGFRβ (Tyr751) (clone 88H8) (Cell Signaling); polyclonal anti-rabbit or -mouse IgG (GE Healthcare, Chicago, IL) was the secondary antibody.

    Techniques: Immunohistochemistry

    PDGFRβ expression in tumor according with expression of CDCP1 protein (IHC CDCP1) and CDCP1 genetic gain

    Journal: BMC Cancer

    Article Title: The PDGFRβ/ERK1/2 pathway regulates CDCP1 expression in triple-negative breast cancer

    doi: 10.1186/s12885-018-4500-9

    Figure Lengend Snippet: PDGFRβ expression in tumor according with expression of CDCP1 protein (IHC CDCP1) and CDCP1 genetic gain

    Article Snippet: Biochemical analyses were performed using rabbit polyclonal antibodies against CDCP1, phospho-CDCP1 (Tyr734), p44/42 MAPK (ERK1/2), phospho-p44/42 MAPK (ERK1/2) (Thr202/Tyr204) (Cell Signaling, Danvers, MA), and PDGFRβ (Santa Cruz Biotechnology, Dallas, TX) or mouse monoclonal anti-phospho-PDGFRβ (Tyr751) (clone 88H8) (Cell Signaling); polyclonal anti-rabbit or -mouse IgG (GE Healthcare, Chicago, IL) was the secondary antibody.

    Techniques: Expressing