A cDNA for WT Parkin was from GeneCopoeia. E3 ligase loss-of-function Parkin Ser65Ala (S65A) and Cys431Ser (C431S) mutants were generated using PfuUltra II fusion HS DNA Polymerase (Agilent Technologies). Ub-defective K2 mutant Lys581Ala (K581A), Lys 582Ala (K582A), or DM Lys581Ala/Lys582Ala (K581A/K582A) were generated using Stratagene QuikChange II XL Site-Directed Mutagenesis kit (Agilent Technologies) and confirmed by DNA sequencing. Protein expression was assessed by Western blotting and visualized by chemiluminescence (26 ). In vitro Ub studies were carried out as described (37 (link)).
Yeon M., Bertolini I., Agarwal E., Ghosh J.C., Tang H.Y., Speicher D.W., Keeney F., Sossey-Alaoui K., Pluskota E., Bialkowska K., Plow E.F., Languino L.R., Skordalakes E., Caino M.C, & Altieri D.C. (2023). Parkin ubiquitination of Kindlin-2 enables mitochondria-associated metastasis suppression. The Journal of Biological Chemistry, 299(6), 104774.
CdnaChemiluminescence Dna polymerase E3 ligase Parkin Protein Site directed mutagenesis
Corresponding Organization : The Wistar Institute
Other organizations :
Case Western Reserve University, Cleveland Clinic Lerner College of Medicine, Sidney Kimmel Cancer Center, Thomas Jefferson University, University of Colorado Denver
Protein expression assessed by Western blotting and visualized by chemiluminescence
In vitro Ub studies
control variables
Wild-type (WT) Parkin
controls
Positive control: Not specified
Negative control: Not specified
Annotations
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