Compound 68 and Fx2149 Biochemical Assays

Compound 68 was ordered through Chembridge and Fx2149 was designed/synthesized by Dr. Fengtian Xue’s group using 68 as a lead compound (Li et al., 2014 (link);Li et al., 2015 (link)). Both compounds were dissolved in 0.1% DMSO for use in biochemical assays. Anti-Flag antibodies, γ-tubulin antibody, anti-Flag-agarose, GTP-agarose, and PR-619 (deubiquitinase inhibitor) were purchased from Sigma (St. Louis, MO, USA). Anti-HA antibody (clone Y-11) was purchased from Santa Cruz (Dallas, TX, USA). Anti-ubiquitin antibody (clone P4D1) was purchased from Covance (Princeton, NJ, USA). Alexa Fluor 568 goat anti-rabbit antibody, LipofectAMINE and Plus transfection reagents, and DMEM cell culture media were from Invitrogen™, and FITC goat anti-mouse antibody was from EMD Millipore (Billerica, MA, USA).

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Thomas J.M., Wang X., Guo G., Li T., Dai B., Nucifora L.G., Nucifora FC J.r., Liu Z., Xue F., Liu C., Ross C.A, & Smith W.W. (2020). GTP-binding Inhibitors Increase LRRK2-linked Ubiquitination and Lewy Body-like Inclusions. Journal of cellular physiology, 235(10), 7309-7320.

Publication 2020

Anti-Flag antibodies γ-tubulin antibody anti-Flag-agarose GTP-agarose PR-619 Alexa Fluor 568 goat anti-rabbit antibody DMEM cell culture media

Agarose Alexa 568 Anti antibodies Anti antibody Antibody Assays Cell Cell culture Clone Culture media Deubiquitinase Dmso Fitc Goat Gtp agarose Lipofectamine Mouse Pr 619 Rabbit Transfection Tubulin Ubiquitin

Corresponding Organization : Johns Hopkins University

Other organizations : University of Maryland, Baltimore, Biogen (United States), Soochow University

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Variable analysis

independent variables

Compound 68 (ordered through Chembridge)
Fx2149 (designed/synthesized by Dr. Fengtian Xue's group using 68 as a lead compound)

dependent variables

Biochemical assay outcomes

control variables

0.1% DMSO (used to dissolve both compounds)
Anti-Flag antibodies, γ-tubulin antibody, anti-Flag-agarose, GTP-agarose, and PR-619 (deubiquitinase inhibitor) (purchased from Sigma)
Anti-HA antibody (clone Y-11, purchased from Santa Cruz)
Anti-ubiquitin antibody (clone P4D1, purchased from Covance)
Alexa Fluor 568 goat anti-rabbit antibody, LipofectAMINE and Plus transfection reagents, and DMEM cell culture media (from Invitrogen™)
FITC goat anti-mouse antibody (from EMD Millipore)

positive controls

Not explicitly mentioned

negative controls

Not explicitly mentioned

Annotations

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