Journal: BMC Cancer

Article Title: CUB Domain Containing Protein 1 (CDCP1) modulates adhesion and motility in colon cancer cells

doi: 10.1186/1471-2407-14-754

Figure Lengend Snippet: CDCP1 protein expression in colon cancer cell lines and modulation of adhesion and motility. A) Cell-surface expression of CDCP1 was measured by flow cytometry using monoclonal mouse anti-CDCP1 clone CUB1. Bound primary antibody was detected with Alexa488-conjugated goat anti-mouse IgG. Standard error bars are shown. n ≥3, except HaCaT where n =2. Cell lines are indicated that showed significantly different CDCP1 cell-surface expression from HaCaT as determined by independent sample 2-tailed t-tests not assuming equal variance. GMF CDCP1, geometric mean fluorescence of bound anti-CDCP1. *p ≤0.05; *** = p ≤0.001. B) Western blot for CDCP1. Total cell protein (40 μg) was separated by SDS-PAGE, transferred to PVDF and CDCP1 detected using 1 μg/ml goat polyclonal antibody anti-CDCP1 antibody (ab1377). The membrane was re-probed for GAPDH to assess protein loading. The image is representative of three blots from independent lysates. C) Binding of stable CDCP1-expressing Colo320 (Colo320-CDCP1) and control Colo320 (Colo320-pcDNA3) cells to control or 10 μg/ml Matrigel coated tissue culture plates. The Colo320 cell-substratum assays were repeated four times. *** = p ≤0.001. D) Adhesion of SW480 cells to control or Matrigel-coated tissue culture plates following reduction of CDCP1 by RNA interference. The experiment was repeated three times. Control, cells transfected with control siRNA; CDCP1, cells transfected with CDCP1 siRNA oligo 2 (see Additional file : Figure S2). E) Motility of SW480 cells through an 8 μm pore membrane towards serum following reduction of CDCP1 by RNA interference. This was measured in arbitrary units (AU) at 60 h after siRNA transfection using the xCELLigence system Real-Time Cell Analyzer Dual Plate (RTCA DP, Roche). UT, untransfected cells; mock, cells treated with Lipofectamine 2000 only; Control siRNA, cells transfected with CDCP1 siRNA oligo 2. See Additional file : Figure S3 for a more extensive kinetic analysis.

Article Snippet: Goat polyclonal anti-CDCP1 (Ab1377) was from Abcam (Cambridge, U.K.).

Techniques: Expressing, Flow Cytometry, Fluorescence, Western Blot, SDS Page, Binding Assay, Transfection

Journal: BMC Cancer

Article Title: CUB Domain Containing Protein 1 (CDCP1) modulates adhesion and motility in colon cancer cells

doi: 10.1186/1471-2407-14-754

Figure Lengend Snippet: CDCP1 and CD9 expression in colon cancer cell lines. A) Cell-surface CD9 expression was measured by flow cytometry using monoclonal mouse anti-CD9 clone ALB6. The primary antibody was detected with Alexa488-conjugated goat anti-mouse IgG. Ten thousand cells were gated per sample. Standard error bars are shown. GMF, geometric mean fluorescence. B) Western blot analysis of CD9 expression. Total cell protein (40 μg) in RIPA buffer was separated by SDS-PAGE, transferred to PVDF and CD9 detected using 2 μg/ml mouse monoclonal antibody clone 602–29. Subsequently the PVDF membrane was stripped and re-probed for GAPDH to assess loading. The image is representative of three blots. Arrows indicate the locations of the CD9 species termed p27 and p25. C) An XY plot of the average CDCP1 and CD9 cell surface expression per cell line. A line of best fit was generated by linear regression. There is a significant positive correlation by bivariate Pearson correlation analysis. *p =0.044. GMF, geometric mean fluorescence. D) CDCP1 and CD9 mRNA expression determined by microarray (Robust multi-array average (RMA)) from 47 colon cancer cell lines was downloaded from the Cancer Cell Line Encyclopedia . An XY plot was generated. The cell lines that were experimentally tested for cell-surface CDCP1 and CD9 protein expression are highlighted in red. A line of best fit was generated by linear regression. There is a highly significant positive correlation as reported by a bivariate Pearson correlation analysis. ***p < 0.001.

Article Snippet: Goat polyclonal anti-CDCP1 (Ab1377) was from Abcam (Cambridge, U.K.).

Techniques: Expressing, Flow Cytometry, Fluorescence, Western Blot, SDS Page, Generated, Microarray

Journal: BMC Cancer

Article Title: CUB Domain Containing Protein 1 (CDCP1) modulates adhesion and motility in colon cancer cells

doi: 10.1186/1471-2407-14-754

Figure Lengend Snippet: Analysis of CDCP1 and CD9 in detergent-resistant membrane (DRM) fractions of HT-29 colon cancer cells. HT-29 cells were plated at 3.8 × 10 6 cells per T175 flask and grown to confluence. The cells were scraped into MBS containing either 0.5% Triton-X100, Brij58 or Brij97 on ice. The lysates were fractionated by sucrose density gradient centrifugation as described in Materials and Methods. Eleven 0.5 ml fractions were collected, with fraction 1 being the top and fraction 11 the bottom of the gradient. The fractions were analysed by Western blotting using the following antibodies (final concentrations in parentheses): CDCP1 (1 μg/ml goat ab1377), CD9 (2 μg/ml mouse monoclonal 602–29), TfR (100 ng/ml mouse monoclonal H68.4 (13–6800)), Flotillin-1 (50 ng/ml). A, B: Triton X-100 extraction; C, D: Brij58 extraction; E, F: Brij97 extraction. B, D, F: Quantitation of the Western blots shown in panels A,C and E respectively using Advanced Image Data Analyzer (AIDA) software.

Article Snippet: Goat polyclonal anti-CDCP1 (Ab1377) was from Abcam (Cambridge, U.K.).

Techniques: Gradient Centrifugation, Western Blot, Quantitation Assay, Software

Journal: BMC Cancer

Article Title: CUB Domain Containing Protein 1 (CDCP1) modulates adhesion and motility in colon cancer cells

doi: 10.1186/1471-2407-14-754

Figure Lengend Snippet: Co-immunoprecipitation of CDCP1 and CD9. SW480 cells were lysed in 1% (w/v) Brij97, 10 mM Tris–HCl (pH 7.4), 150 mM NaCl, 1 mM CaCl 2 , 1 mM MgCl 2 . A. Immunoprecipitation from whole cell extracts of SW480 cells was performed using 5 μg of ALB6 anti-CD9 monoclonal antibody (lane 6) or isotype-matched control antibody (lane 5). Immunoprecipitates were analysed by Western blotting with antibodies against either CDCP1 (ab1377), top panel; EpCAM (ab32392), middle panel or CD9 (602.29), lower panel. Further controls for the specificity of immunoprecipitation and Western blotting are: lane 1: 15 μg whole cell extract, lanes 2 and 3: 5 μg of isotype-control antibody and anti-CD9 antibody respectively; lane 4: SW480 proteins bound to protein G beads. B. Densitometric analysis of the Western blot for CDCP1. A portion of the blot corresponding to the CDCP1 p135 region was analysed in the tracks generated by immunoprecipitation with the control (red) and anti-CD9 (green) antibodies using AIDA software.

Article Snippet: Goat polyclonal anti-CDCP1 (Ab1377) was from Abcam (Cambridge, U.K.).

Techniques: Immunoprecipitation, Western Blot, Generated, Software