Journal: Journal of medicinal chemistry

Article Title: Chemical Toolkit for PARK7: Potent, Selective, and High-Throughput.

doi: 10.1021/acs.jmedchem.2c01113

Figure Lengend Snippet: Figure 2. Selectivity analysis of 8RK59 toward UCHL1 and PARK7 and structural characterization of 8RK64 binding to PARK7. (A) Structures of 8RK64 and 8RK59. (B) Labeling of PARK7 and UCHL1 with 8RK59. UCHL1 and PARK7 were incubated with 8RK59, resolved by SDS-PAGE, and analyzed by fluorescence scanning (top) and Coomassie staining (bottom). DMSO and BodipyFL were used as negative controls. (C) Co- crystal structure of the 8RK64-PARK7 complex (PDB: 7PA2). PARK7 in green and 8RK64 in cyan.

Article Snippet: The fluorescence intensity (FI) signal was monitored with a BMG Labtech PHERAstar plate reader (λex/em 350/450 nm) for 1 h. All samples were normalized to the positive and negative controls and plotted against the inhibitor concentrations (in μM) using the built-in equation “[inhibitor] vs response − variable slope (four parameters), least-squares fit” with constraints “Bottom = 0” and “Top = 100” in GraphPad Prism 9.0.1 software to obtain the IC50 values.

Techniques: Binding Assay, Labeling, Incubation, SDS Page, Fluorescence, Staining

Journal: Journal of medicinal chemistry

Article Title: Chemical Toolkit for PARK7: Potent, Selective, and High-Throughput.

doi: 10.1021/acs.jmedchem.2c01113

Figure Lengend Snippet: Figure 4. Biochemical characterization of the fluorescent PARK7 probes. (A) Labeling efficiency of JYQ-92, JYQ-93, and JYQ-107 with purified PARK7. PARK7 was incubated with increasing concentrations of the probes. (B) JYQ-92 labels purified recombinant PARK7 WT but not Cys106Ser mutant. (C) Fluorescence labeling of PARK7 activity in the HEK293T cell lysate. The pink arrow indicates the band for PARK7. (D) Fluorescence labeling by JYQ-92 in the HEK293T cell lysate with/without depletion of PARK7 or UCHL1.The prepared cell lysate was incubated with JYQ-92 (1 μM final concentration) for 1 h. (E) Fluorescence labeling of PARK7 remaining activity by JYQ-92 after treatment with inhibitor JYQ-88. The prepared cell lysate was incubated with a dilution series of JYQ-88, followed by incubation with JYQ-92 (1 μM). HEK293T cell lysates treated as indicated above were analyzed by immunoblotting against total UCHL1 (rabbit anti-UCHL1 antibody, 1:1000) and PARK7 (rabbit anti-PARK7 antibody, 1:1000), with actin (mouse anti-actin antibody, 1:10,000) as the loading control. Relevant antibodies used in each gel are indicated using the “α” symbol in front of the protein name.

Article Snippet: The fluorescence intensity (FI) signal was monitored with a BMG Labtech PHERAstar plate reader (λex/em 350/450 nm) for 1 h. All samples were normalized to the positive and negative controls and plotted against the inhibitor concentrations (in μM) using the built-in equation “[inhibitor] vs response − variable slope (four parameters), least-squares fit” with constraints “Bottom = 0” and “Top = 100” in GraphPad Prism 9.0.1 software to obtain the IC50 values.

Techniques: Labeling, Purification, Incubation, Recombinant, Mutagenesis, Fluorescence, Activity Assay, Concentration Assay, Western Blot, Control