Proximity based proteomics identifies the association of UCH-L1 with translation initiation factors. (A) Schematic of the BioID2 constructs. Myc = Myc epitope tag. (B) The expression of the BioID2-UCHL1 fusions in KMS-11 cells was determined by immunoblot, as shown. Compared with the endogenous UCH-L1 (molecular weight, ∼25 kDa). Catalytic activity of the endogenous and BioID2 fusion was assessed by reactivity with the activity-based probe ubiquitin vinylmetrhyl esther (UbVME). Activity is indicated by a shift in mass of approximately 8 to 10 kDa resulting from the covalent adduct formed with active enzymes and UbVME. (C) The functional integrity of the BioID fusion proteins was assessed by its ability to promote phosphorylation of AKT and to suppress that of S6 kinase. Expression of UCHL1-HA was used for comparison. The relevant region of the blots was cropped to show the expression of BioID2-UCH-L1, or UCHL1-HA. (D) UCH-L1 (green) immunostaining of HeLa cells expressing UCHL1-HA or BioID2-UCHL1. Localization was also examined through the Myc-tag on BioID2 (red). Where indicated, biotin was added to cells expressing BioID-UCHL1 and labeled proteins were visualized with avidin (red). Scale bar = 10 μm.