Fig. 6

Characterization of CDCP1-containing protein complexes reveals that MMP14 preferentially associates with cleaved CDCP1, while Galectin 1 only binds to full-length CDCP1. a Coomassie-stained SDS-PAGE gel of proteins affinity purified from MDA-MB-231 cells transduced with FLAG-tagged ΔCDCP1 or CDCP1 and treated for 15 minutes with or without the tyrosine phosphatase inhibitor sodium pervanadate (100 μM) to elevate overall tyrosine phosphorylation. Protein bands were excised and identified by mass spectrometry as described in the “Methods” section. b Extracts from MDA-MB-231 cells transduced with the indicated adenoviral vectors were affinity purified with anti-FLAG agarose to isolate CDCP1-containing complexes and the purified material or corresponding crude lysates were analyzed by immunoblot. c Study performed as in Fig. 5b except that cells were treated for 24 hours with either vehicle, 20 μM of the matrix metalloproteinase inhibitor GM6001 or 100 nM of the Src-family kinase inhibitor dasatinib. d Silver-stained SDS-PAGE gel of proteins isolated by anti-FLAG affinity purification from MDA-MB-231 cells transduced with the indicated adenoviruses and treated for 15 minutes with or without 100 μM sodium pervanadate. A low-molecular-mass protein was observed (red arrow) that was present when full-length forms of CDCP1 were expressed, but not when cleaved forms of CDCP1 (ΔCDCP1) were expressed. e HEK 293 cells were transiently transfected with the indicated vectors and CDCP1 or ΔCDCP1-containing complexes were isolated with anti-FLAG agarose and analyzed by immunoblot