Fig. 4: ProtacID identifies a non-productive PROTAC-protein interaction.

a Dot Plot (dot attributes as in Fig. 2d) highlighting KIF20B, SMARCA2, and SMARCA4 identification in ProtacID analyses (293 cells) with the indicated PROTAC, or standard BioID analysis. n = 3 biological replicates. b 293 cells expressing FmT-ΔVHL were treated with increasing concentrations (0, 10, 100, 1000 nM) of the indicated PROTACs, biotin was added, and streptavidin pulldowns were conducted (as in Fig. 2c). Isolated proteins (pulldown, PD) or whole cell lysates (input) were subjected to western blotting with the indicated antibodies. n = 3 biological replicates. c Global proteomics analysis of ACBI1-treated 293 Flp-In cells. Cells were treated for 3 h with DMSO or ACBI1 (1 μM, n = 5 biological replicates/condition). Five hundred nanograms of whole cell lysate were analyzed on a Bruker TIMS-TOF mass spectrometer in DIA mode. Differential abundance analysis was performed using linear modeling with empirical Bayes moderation (limma), applying a two-sided moderated t-test with Benjamini-Hochberg multiple testing correction. d (left) Dot Plot highlighting KIF20B, SMARCA2 and SMARCA4 identifications in an ACBI1 ProtacID experiment conducted in the absence of the neddylation inhibitor MLN4924. (Dot attributes as indicated in Fig. 2d.) (right) Western blotting results of the same experiment (ACBI1 gradient 0, 10, 100, 1000 nM). n = 3 biological replicates. e ProtacID workflow for the identification of productive and non-productive PROTAC-protein interactions. ProtacID conducted in the presence of a PROTAC and MLN4924 can identify both productive and non-productive PROTAC interactors (along with their interacting protein partners). ProtacID conducted in the presence of a PROTAC, but in the absence of MLN4924, will identify only non-productive PROTAC interactors. Source data are provided as a Source data file.