Fig. 3. USP30 activity profiling using IMP-2587 and IMP-2586 in HEK293 cells. (a) Affinity pulldown and chemical proteomics workflow. Cells were incubated with IMP-2587 and IMP-2586 for 1 hour, separately and labelled proteins ligated via CuAAC to AzRB or AzT for IMP-2587 and YnB or YnT for IMP-2586 for in-gel fluorescence and/or affinity enrichment for immunoblotting and proteomics. Total cell lysates are labelled as TL samples; supernatants following affinity enrichment were collected as SN samples; protein bound to the beads was collected as PD samples. (b) In-gel fluorescence shows dose-dependent labelling by IMP-2587. (c) Probe-labelled (IMP-2587) protein identification by enrichment and immunoblotting for USP30. (TL: Total Lysate, SN: Supernatant, PD: Pull-Down) (d) In-gel fluorescence shows dose-dependent labelling by IMP-2586. (e) Probe-labelled (IMP-2586) protein identification by enrichment and immunoblotting for USP30.