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: Images were captured using a chemiluminescence imager (Fuji LAS-3000, Japan) and analysed using AIDA software (Raytek Scientific Ltd, Sheffield, UK).

Techniques: Membrane, Gradient Centrifugation, Western Blot, Extraction, 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: Images were captured using a chemiluminescence imager (Fuji LAS-3000, Japan) and analysed using AIDA software (Raytek Scientific Ltd, Sheffield, UK).

Techniques: Immunoprecipitation, Control, Western Blot, Generated, Software