Probing UCHL1 activity
in cells with 8RK59. (A) Schematic
overview of labeling UCHL1 activity with 8RK59 in cells
with/without the depletion of UCHL1 or PARK7 and subsequent assays
that were performed to characterize the staining specificity. (B)
Live-cell fluorescence imaging of the 8RK59-labeled control
(PLKO), 6RK73 (PLKO pretreated with 6RK73), and shUCHL1
and shPARK7 A549 cells. (C) Western blotting (WB) of UCHL1 in the
control, 6RK73, and shUCHL1 and shPARK7 A549 cells. WB
for GAPDH was included as a loading control. (D) WB of PARK7 in the
control, 6RK73, and shUCHL1 and shPARK7 A549 cells. WB
for GAPDH was included as a loading control. (E) DUB activity assay
of the control, 6RK73, and shUCHL1 and shPARK7 A549 cells
with Rh-Ub-PA. The UCHL1-Ub band is indicated with an arrow. (F) Fluorescence
scanned SDS-PAGE gel of the 8RK59-labeled control, 6RK73, and shUCHL1 and shPARK7 A549 cells. UCHL1 and PARK7
bands are indicated with arrows. (G) Immunofluorescence (IF) staining
of UCHL1 in an 8RK59-labeled control and shUCHL1 A549
cells. A 10 μm scale bar is included. Squares indicate areas
that were used for close-up images. All cells from B to G were treated
with 5 μM 8RK59 overnight, and 6RK73 group cells were pretreated with 5 μM 6RK73 for
4 h.