FIGURE 7. UCH37 Potentiates Proteasomal Degradation in Cells.

(A) Steady-state levels of Ub conjugates were assessed in WT and UCH37^KO cells using the indicated antibodies. WT and UCH37^KO cells were transfected with inactive (C88A and C88S) and active forms of UCH37 and the levels of Ub conjugates were also probed.

(B) Schematic for GFP^u turnover in cells.

(C) Fluorescence histograms of WT HEK293 GFP^u cells, UCH37^KO GFP^u cells, and UCH37^KO GFP^u cells expressing either WT UCH37 (UCH37^KO + WT UCH37) or inactive UCH37 (UCH37^KO + UCH37 C88S). Mean GFP fluorescence measured in 2 independent experiments.

(D) Schematic for measuring global protein turnover using azidohomoalanine (AHA) labeling of newly synthesized proteins in cells. AHA incorporated proteins were labeled with DBCO-Cy5, separated by SDS-PAGE and visualized by Cy5 fluorescence.

(E) Cy5 fluorescence analysis of the turnover of AHA-labeled proteins at different chase times in WT, UCH37^KO, and UCH37^KO HEK293 FT cells expressing WT UCH37 or inactive UCH37 C88S (top) with corresponding Sypro total protein stain (bottom).

(F) Densitometric quantification of AHA labeled proteins with molecular weights ≤ 50 kDa. Values at each time point are adjusted relative to total protein. Results are shown as mean ± SEM from 3 independent experiments.

(G) Model showing UCH37-catalyzed Ub chain debranching promotes proteasomal degradation.

See also Figure S7 and Table S1H.