Figure 2S: Specificity of the Anti-Phospho-CDCP1 Antibodies

Supplementary Information:

Figure 1S: Correlation matrix of the proteins that showed phospho-tyrosine signal in more than 50% of the samples included in the study.

Figure 2S: Specificity of the anti-phospho-CDCP1 antibodies:

CDCP1 WT or mutated at tyrosine 734 or 743 as indicated was expressed in U2OS cells together with Src or by itself, and anti-phospho-CDCP1 antibodies used to detect the state of CDCP1 phosphorylation. Left panel: the phospho-CDCP1 antibodies are site specific. Right panel: the phospho-CDCP1 antibodies are phospho-specific.

Figure 3S:CUB1 mAb promotes the association of CDCP1 with SRC in MCF10A cells. Cells were grown in DMEM/10% FBS to near confluence and serum-starved overnight in DMEM/0.1% FBS. After exposure of cells to CUB1 mAb (0.5 ug/ml) for 30 min, cells were lysed. The lysates were subjected to immunoprecipitation overnight with antibodies to various SFK proteins, including SFK (SC-18, 2 ug/ml), YES (SC-14, 2 ug/ml), SRC (CST2123, 4 ul/ml), and FYN (SC-434, 2 ug/ml). The immunoprecipitates were subjected to SDS-PAGE and Western blotting using the indicated antibodies. The SRC and P-416 SRC immunoblots present film exposures for both a short and long (lg) time. Exposure of cells to CUB1 mAb promoted the association of phosphorylated CDPC1 protein with SRC, SFK, and YES proteins. The modest amount of activated CDCP1 in the immunoprecipitation by the anti-FYN antibody was due to the association of CDCP1 with the CUB1 mAb itself, and was similar to that observed using antibodies to LYN and LCK (not shown).

Figure 4S: Time Course of the dephosphorylation of FAK in response to CDCP1 activation. HCT116 cells were treated for the indicated time in conditions of attachment (as used for culture maintenance) and 0.1% FBS. The phosphorylation of FAK and CDCP1 was determined by Western blotting as indicated.

Figure 5S. The inhibition of FAK activity increases the phosphorylation of CDCP1 on Tyr734 in MCF10A cells. Cells were grown in DMEM/10% FBS to near confluence. Cells were then serum-starved overnight in DMEM/0.1% FBS and exposed to CUB1 mAb (0.5 ug/ml) for 30 min or the FAK inhibitor PF573228 (10 uM) for 30-90 min. Cell lysates were subjected to SDS-PAGE and Western blotting using the indicated antibodies. PF573228 blocked the phosphorylation of FAK on Tyr397 and produced an increase in the phosphorylation of CDCP1 on Tyr734 as early as 30 min.

Figure 6S:CUB1 mAb-stimulated migration of MCF10A cells is dependent on Src activity. Cell migration (from top to bottom of filter) was measured in the absence and presence of CUB1 (0.5 ug/ml) and the Src inhibitor SKI-606 (Panel A, 1 uM) or the FAK inhibitor PF573228 (Panel B, 1uM). *P<0.05, n=3.

Figure 7S: P-Tyr311 PKCd is Found at Cell-Cell Junctions Upon CUB1 Stimulation.

MCF10A Cells were stimulated with CUB1 mAb for 60 minutes. Top Panels: P-Tyr311 was detected with a specific antibody. DAPI was used for nuclei stain. CUB1 (used for stimulation) labels CDCP1 at cell-cell contacts (bottom panel).

Figure 8S: CUB1 induces faster formation of calcium dependent cell-cell junctions.

MCF10A cells were cultured overnight in low calcium conditions (SMEM). Culture medium was switched to DMEM (Calcium >1 mM) for 30 minutes or kept in SMEM. CUB1 was added at the time of medium change where indicated. Nascent calcium dependant cell-cell junctions were detected using an anti beta-catenin antibody.

Figure 9S: Increased Tyr311 PKC phosphorylation induced by co-treatment with PMA and CUB1. Cell stimulation was performed for 10 minutes with the indicated stimuli in the presence or absence of CUB1 (0.5 ug/ml). Total ERK blot is used as a loading control. Insulin (Ins, 100 nM), PMA (100 nM), Carbachol (Carb, 10 uM), IGF1 (100 nM), EGF (100 nM), LPA (5 uM), PDGF ( 5 ng/ml), UTP (100 uM).

Figure 10S: Co-stimulation with PMA increases the effects of CUB1 on cell-cell contacts.

Cells were stimulated for 30 minutes with the indicated stimuli (CUB1, 0.5 ug/ml and PMA, 100 nM). Actin staining was performed using rhodamine phalloidin (red). Cell-cell contacts were characterized by staining adherens junctions using an anti-beta-catenin antibody (green). Nuclei were stained with DAPI (blue). Top panel: Actin stain. Bottom panel: All channels merged.

Table 1S Differentially phosphorylated proteins between normal and cancer
Protein ID / logFC / Average Phospho-counts / t* / P.Value / adj.P.Val† / B‡
PRP4 / -1.2814 / 1.700143 / -10.792 / 9.30E-21 / 4.90E-18 / 33.36216
GSK3A / -0.69512 / 3.181351 / -7.85894 / 4.80E-13 / 1.27E-10 / 15.67778
AFAP / 0.856415 / 0.107052 / 72.16817 / 1.20E-10 / 1.27E-08 / 15.57402
DYRK1B / -1.03262 / 1.66463 / -7.71589 / 1.10E-12 / 1.93E-10 / 14.85897
HIPK3 / -0.89655 / 0.663781 / -7.70653 / 1.90E-12 / 2.50E-10 / 14.41477
HSPA2 / 1.121563 / 0.160223 / 86.00793 / 8.94E-10 / 6.73E-08 / 13.66257
DYRK1A / -0.93774 / 1.828 / -6.51299 / 8.57E-10 / 6.73E-08 / 8.282754
HIPK1 / -0.85575 / 1.232894 / -6.3254 / 2.89E-09 / 1.65E-07 / 7.178848
HIPK2 / -0.85575 / 1.232894 / -6.3254 / 2.89E-09 / 1.65E-07 / 7.178848
STAT5A / -0.41047 / 0.27778 / -6.28152 / 3.13E-09 / 1.65E-07 / 7.035321
STAT5B / -0.41524 / 0.291052 / -6.24707 / 3.74E-09 / 1.79E-07 / 6.861484
CD3D / 1.52582 / 0.381455 / 73.53497 / 3.47E-05 / 0.001104975 / 3.83517
K14 / -1.50862 / 0.377155 / -72.7061 / 3.56E-05 / 0.001104975 / 3.810056
FAM62A / -0.49007 / 0.408165 / -5.56649 / 1.14E-07 / 5.02E-06 / 3.534051
K16 / -1.22926 / 0.310204 / -57.4659 / 6.37E-05 / 0.001766408 / 3.245046
septin 9 / 1.121563 / 0.280391 / 54.05231 / 7.41E-05 / 0.001951734 / 3.085286
Fes / 0.942812 / 0.235703 / 45.43762 / 0.000114 / 0.002852268 / 2.60804
KCTD7 / 1.275581 / 0.637791 / 31.50588 / 0.00028 / 0.005680876 / 1.407246
GRF-1 / -0.616 / 1.460294 / -4.92485 / 2.05E-06 / 8.31E-05 / 0.677573
PRPF31 / 1.002238 / 0.334079 / 35.12307 / 0.003995 / 0.042967707 / -0.62254
TDRD6 / -0.94834 / 0.711287 / -16.1175 / 0.001456 / 0.018269953 / -0.67723
Sin3A / -0.92079 / 0.736635 / -10.1499 / 0.001063 / 0.01514016 / -1.11922
ERK2 / -0.47413 / 1.190479 / -4.37602 / 2.19E-05 / 0.000825038 / -1.59117
Src / -0.60222 / 1.167865 / -4.28654 / 3.35E-05 / 0.001104975 / -1.91301
ERP29 / 1.191746 / 0.794497 / 7.119524 / 0.001339 / 0.017320335 / -2.1422
SAP97 / 0.957564 / 0.522307 / 4.461443 / 0.000228 / 0.004800182 / -2.42221
Lck / -0.59249 / 1.426224 / -4.13362 / 5.93E-05 / 0.001735893 / -2.50617
Brk / -0.56137 / 0.367877 / -4.03616 / 0.000155 / 0.00371731 / -2.90347
ALK / 2.920775 / 1.982526 / 4.696719 / 0.004252 / 0.043939824 / -3.564
Eps8 / -0.66208 / 0.529022 / -3.82189 / 0.000311 / 0.006077251 / -3.56898
CDK3 / -0.59284 / 1.540069 / -3.84707 / 0.000173 / 0.003970453 / -3.56954
ANXA2 / 0.674879 / 1.980426 / 3.816541 / 0.000194 / 0.004253034 / -3.66584
CD46 iso4 / -0.71622 / 0.504777 / -3.71809 / 0.000539 / 0.009469869 / -3.91231
CDK2 / -0.57868 / 1.559229 / -3.63845 / 0.000371 / 0.006989219 / -4.29109
FRK / -0.60371 / 0.538336 / -3.53624 / 0.000717 / 0.011932046 / -4.4224
ERK1 / -0.41062 / 1.085039 / -3.56066 / 0.000492 / 0.008941008 / -4.5407
ERK5 / -0.42157 / 0.440157 / -3.51313 / 0.000747 / 0.011932046 / -4.55434
ERK5 iso2 / -0.42157 / 0.440157 / -3.51313 / 0.000747 / 0.011932046 / -4.55434
Fgr / 0.879112 / 0.824046 / 3.44536 / 0.000971 / 0.014380541 / -4.68963
KIAA1217 / -0.18041 / 0.066629 / -3.46165 / 0.000841 / 0.013029186 / -4.69607
Cas-L / 0.768829 / 1.047559 / 3.41328 / 0.001206 / 0.016290771 / -4.80925
CDCP1 / -0.69604 / 1.015284 / -3.33132 / 0.001348 / 0.017320335 / -5.09415
p38-alpha / -0.56374 / 3.220136 / -3.35826 / 0.000982 / 0.014380541 / -5.20473
calmodulin / 0.289137 / 0.286014 / 3.24605 / 0.001546 / 0.018942813 / -5.37822
IQGAP1 / -0.45241 / 0.300418 / -3.18814 / 0.002937 / 0.032929021 / -5.38356
STAT3 / 0.302557 / 2.136134 / 3.299468 / 0.001188 / 0.016290771 / -5.39832
p38-gamma / -0.44185 / 0.71798 / -3.10285 / 0.002483 / 0.028447319 / -5.77598
PDHA1 / 0.548062 / 1.519965 / 3.111694 / 0.002345 / 0.027457104 / -5.81109
PDHA2 / 0.548062 / 1.519965 / 3.111694 / 0.002345 / 0.027457104 / -5.81109
tensin 1 / 0.859472 / 2.042103 / 3.016325 / 0.003313 / 0.036369885 / -6.01
tensin 2 / 0.358961 / 0.464961 / 2.923424 / 0.004131 / 0.043538479 / -6.34263

Note: A negative logFC (log-fold-change) indicates that the spectral counts of the phosphorylated peptides from the respective proteins are higher in cancer than normal samples, while a positive logFC indicates that the latter have higher counts than their cancer counterparts.

*t refers to the moderated t-statistic obtained from the empirical Bayes shrinkage model

†adj.P.Val refers for the adjusted P-values for multiple testing

‡The B-statistic (lods or B) is the log-odds that a protein is differentially phosphorylated.

Table 2S Phosphotyrosine proteins significantly (P<0.1) correlated positively or negatively (Pearson’s correlation) with the phosphorylation of CDCP1 in normal samples (n=40)
Correlation / PTYR Protein / Coefficient / P-value
Positive / Claudin 5 / 0.98 / 2.88E-07
MET / 0.99 / 0.0013
APP / 0.66 / 0.0037
PKCD / 0.47 / 0.0047
PTRF / 0.76 / 0.0064
ERK5 / 0.55 / 0.01
DDR1 / 0.39 / 0.0189
HIPK2 / 0.37 / 0.0196
ANXA2 / 0.36 / 0.0234
HIPK3 / 0.36 / 0.0246
p38-delta / 0.34 / 0.0315
HBA1 / 0.37 / 0.0374
HRS / 0.53 / 0.0434
ARP3 / 0.64 / 0.045
RA70 / 0.76 / 0.0465
eEF1A-1 / 0.39 / 0.0531
AXL / 0.52 / 0.0549
Actin, alpha 1 / 0.61 / 0.0592
eEF1A-2 / 0.35 / 0.0763
FAM62A / 0.28 / 0.0835
YES / 0.28 / 0.0858
EphA2 / 0.47 / 0.0899
GRF-1 / 0.26 / 0.106
SYK / 0.38 / 0.1061
LCK / 0.26 / 0.1083
Negative / CLH-17 / -0.52 / 0.0226
ACK / -0.59 / 0.0924
SHP-1 / -0.43 / 0.0958
RAIG1 / -0.31 / 0.0971
Tensin 2 / -0.28 / 0.1027
Table 3S Phosphotyrosine proteins significantly (P<0.1) correlated positively or negatively (Pearson’s correlation) with the phosphorylation of CDCP1 in tumor samples (n=36). * The major peptide corresponding to this correlation value is shared by several SFK members (LCK, FYN, YES and SRC)
Correlation / PTYR Protein / Coefficient / P-value
Positive / PKCD / 0.60 / 1.06E-07
SFK* / 0.45 / 7.97E-05
EphA5 / 0.61 / 0.001
SRC / 0.38 / 0.001
PTRF / 0.70 / 0.002
DSC3 / 0.69 / 0.003
FLNB / 0.85 / 0.003
ITGB4 / 0.49 / 0.006
p38-delta / 0.33 / 0.006
JNK1 / 0.66 / 0.007
JNK3 / 0.66 / 0.007
DSC2 / 0.64 / 0.008
EPS8 / 0.44 / 0.011
ARP3 / 0.63 / 0.011
GRF-1 / 0.27 / 0.017
Plakophilin 1 / 0.55 / 0.018
ERK5 / 0.35 / 0.018
YES / 0.28 / 0.018
Plakophilin 3 / 0.60 / 0.019
FLJ32810 / 0.55 / 0.028
APP / 0.40 / 0.031
EphA2 / 0.43 / 0.034
Desmoplakin 3 / 0.79 / 0.034
K8 / 0.79 / 0.035
Cortactin / 0.90 / 0.036
KIAA1217 / 0.31 / 0.036
FAM62A / 0.24 / 0.041
Nectin 1 / 0.96 / 0.042
AXL / 0.43 / 0.049
BAG3 / 0.57 / 0.054
K14 / 0.94 / 0.058
Laminin receptor 1 / 0.38 / 0.058
GDI2 / 0.56 / 0.059
PRP4 / 0.21 / 0.067
FYN / 0.21 / 0.075
HIPK2 / 0.22 / 0.078
FRK / 0.27 / 0.093
FER / 0.45 / 0.094
Actin, alpha 1 / 0.52 / 0.098
K5 / 0.28 / 0.098
Jak1 / 0.73 / 0.099
IRS-1 / 0.72 / 0.107
Negative / Mucin 1 / -0.46 / 0.018
RANBP2L2 / -0.49 / 0.061
RGPD8 / -0.49 / 0.061
G6PD / -0.34 / 0.101