Affinity pull-down and mass spectrometry-based identification of associated proteins. (A) Workflow adopted to identify CD63 interacting proteins using the BioID approach coupled with mass spectrometry. (B) CD63-BirA constructs were over expressed with or without GFP-LMP1 in HEK293 CD63 CRISPR cells. Biotinylated proteins were pulled-down from the total cell lysate using streptavidin magnetic beads. Bound proteins were eluted using sample buffer + 50 mM biotin and resolved on a 4–20% gel, followed by staining with Coomassie brilliant blue. (C) Protein bands were destained, trypsinized, and identified using liquid chromatography coupled with mass spectrometry (LC-MS/MS). Mass spectrometry files were searched against human database using Proteome Discoverer software and further analyzed using Scaffold program. Venn diagram showing the proteins identified in untransfected, transfected with CD63-BirA, and with CD63- BirA and GFP-LMP1. (D) LMP1 modifies the CD63 interactome. The presence of LMP1 changes the interacting proteins to more strongly interacting or less strongly interacting.