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Flowchart of FCMS to identify upstream kinases. Wild type substrate (SUB) or mutated substrate (mSUB) in BiFC vector Myc-VN155 and human kinase cDNA expression library in HA-VC155 vector are cotransfected to different SILAC cells. Cells are combined, and protein complexes are immunoprecipitated with GFP nanobody. Proteins on beads are reduced, alkylated, and tryptic digested, and the resulting peptide samples are analyzed by LC–MS for both protein identification and quantitation.
