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SUPPLEMENTARY FIGURE LEGENDS
Supplementary Figure S1. Quantitative proteomics identifies high level of Y707 and Y806
phosphorylation from CDCP1 in HCC1954 breast cancer cells
(a) Bar charts representing CDCP1 phosphopeptide intensities detected by LC-MS/MS in six
breast cancer cell lines. Briefly, six human breast cell lines (SUM159, MDA-MB231-LM2,
BT474, HCC1954, AU565, SKBR3) were harvested and lysed. After proteins digestion with
trypsin, phosphotyrosine peptides were enriched from peptide mixtures using a 2 steps procedure
(immunoprecipitation with anti-phosphotyrosine antibodies and TiO2enrichment) and analyzed
by liquid chromatography-tandem mass spectrometry (LC-MS/MS) (LTQ orbitrap Elite,
Thermo) as described. The ion chromatogram for Tyr707 and Tyr806 phosphopeptides were
extracted and area-under-the-curve was used for relative quantitation.
(b) HCC1954 cells treated with Dasatinib (1µM) or Saracatinib (1µM) for 2h. Cell lysates from
HCC1954 cells were collected for an overnight immunoprecipitation of CDCP1 followed by
western blot analysis with anti-phospho-Tyr707, -Tyr734, -Tyr806 CDCP1 specific antibodies
(Cell Signaling) and global anti-phosphotyrosine antibody (4G10, Millipore).
Supplementary Figure S2. CDCP1 overexpression activates SFK and their downstream
effectors in HEK293 cells.
(a) Validation of CDCP1-induced protein tyrosine phosphorylation identified by quantitative
phosphoproteomics. Cell lysates from HEK293 cells infected with indicated viruses were
collected for an overnight immunoprecipitation with anti-PLCG1, anti-SHP2, anti-CTNND1
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(Cell signaling) and anti-ITGB1 (Thermo Scientific) followed by western blot analysis with antiphosphotyrosine antibody (4G10, Millipore) and the respective total antibodies. Immunoblots of
the whole cell lysates show SRC, p-SFK Tyr416, PKCδ, p-PKCδ Tyr311, SHIP2, p-SHIP2
Tyr986/987 levels.
(b) Activity of the different SFK in HEK293 cells overexpressing CDCP1 WT. Cell lysates from
HEK293
cells
infected
with
indicated
viruses
were
collected
for
an
overnight
immunoprecipitation of total SFK (CST1 antibody), SRC, LYN, YES and FYN (antibodies from
Cell Signaling) followed by western blot analysis with pSFK Tyr 416 antibody (Biosource) and
the respective total antibodies.
Supplementary Figure S3. CDCP1 downregulation decreases SFK downstream signaling in
breast and lung cancer cells.
Quantitative phosphoproteomics identifies a list of 18 selected phosphopeptides belonging to Src
kinases downstream modulated upon CDCP1 knock down in SKMES-1, NCI-H2122 and
HCC1954 cancer cells. These cells were harvested and lysed. After proteins digestion with
trypsin, phosphotyrosine peptides were enriched from peptide mixtures using a 2 steps procedure
(immunoprecipitation with anti-phosphotyrosine antibodies and TiO2enrichment) and analyzed
by liquid chromatography-tandem mass spectrometry (LC-MS/MS) (LTQ orbitrap Elite,
Thermo) as described. Label free quantification was performed using Progenesis LC-MS. The
normalized peptide intensities were summed for each unique phosphorylated peptide with
mascot score exceeding 20. The phosphopeptide intensities were used to calculate the log2 fold
change ratios of each unique phosphopeptide of vc versus sh#1 or vc versus sh#2 in SKMES-1,
NCI-H2122 and HCC1954 cancer cells. Gene name, peptide sequence, pY site, phosphopeptide
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intensity corresponding to Log2 ratios are shown.* indicates the unique phosphopeptide
sequence for YES, FYN, SRC, LCK proteins.** indicates the unique phosphopeptide sequence
for LYN and HCK proteins.
Supplementary Figure S4. Examples of different level of CDCP1 expression in solid cancer
cells.
Representative images of CDCP1 IHC staining of control cell lines, demonstrating high,
medium-low or negative membranous staining.
Supplementary Table S1. Phosphopeptide intensity of HEK293 overexpressing CDCP1 with or
without Dasatinib treatment.
Supplementary Table S2. List of protein identified after immunoprecipitation of CDCP1 in
HCC1954 cells treated with DMSO or Dasatinib and in HEK293 overexpressing CDCP1 treated
with DMSO or Dasatinib. Total proteins were extracted and CDCP1 IP was performed overnight
at 4°C. After elution from protein sepharose beads, the proteins were loaded and separated on
SDS-PAGE gel. After coomassie blue staining, gel bands were excised. Proteins were reduced,
alkylated and trypsin digested overnight. Peptides were then analyzed by LC-MS as described in
Figure 1c.
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