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. 2020 Sep 4;142(39):16825–16841. doi: 10.1021/jacs.0c07726

Figure 5.

Figure 5

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.