Journal: Open Biology

Article Title: Phosphorylation of Parkin at Serine65 is essential for activation: elaboration of a Miro1 substrate-based assay of Parkin E3 ligase activity

doi: 10.1098/rsob.130213

Figure Lengend Snippet: PINK1-dependent phosphorylation of Parkin Ser 65 leads to activation of Parkin E3 ligase activity and multi-monoubiquitylation of Miro1. Wild-type (WT) ( a ) but not kinase-inactive (KI) ( b ) PINK1 activates wild-type Parkin E3 ligase activity leading to Miro1 multi-monoubiquitylation, an effect that is blocked by mutant Parkin Ser65Ala (S65A) ( c ). Two micrograms of wild-type or S65A Parkin were incubated with indicated amounts of wild-type or kinase-inactive (D359A) MBP-TcPINK in a kinase reaction (50 mM Tris–HCl (pH 7.5), 0.1 mM ethylene glycol tetra-acetic acid (EGTA), 10 mM MgCl 2 , 0.1% 2-mercaptoethanol and 0.1 mM ATP) for 60 min. The ubiquitylation reaction was then initiated by addition of ubiquitylation assay components (50 mM Tris–HCl (pH 7.5), 0.05 mM EGTA, 10 mM MgCl 2 , 0.5% 2-mercaptoethanol, 0.12 μM human recombinant E1 purified from Sf21 insect cell line, 1 μM human recombinant UbcH7 purified from E. coli , 0.05 mM Flag-ubiquitin (Boston Biochem) and 2 mM ATP) and 2 μg of His-Sumo-Miro1. Reactions were terminated after 60 min by addition of SDS–PAGE loading buffer and resolved by SDS–PAGE. Miro1, ubiquitin, Parkin and PINK1 were detected using anti-SUMO, anti-FLAG, anti-Parkin and anti-MBP antibodies, respectively. Representative of three independent experiments.

Article Snippet: Kinase assays were incubated at 30°C for 60 min followed by addition of ubiquitylation assay components and Mastermix to a final volume of 50 μl (50 mM Tris–HCl (pH 7.5), 0.05 mM EGTA, 10 mM MgCl 2 , 0.5% 2-mercaptoethanol, 0.12 μM human recombinant E1 purified from Sf21 insect cell line, 1 μM human recombinant UbcH7 and 2 μg 6xHis-Sumo-Miro1 (wild-type or point mutants) both purified from E. coli , 0.05 mM Flag-ubiquitin (Boston Biochem) and 2 mM ATP).

Techniques: Activation Assay, Activity Assay, Mutagenesis, Incubation, Ubiquitin Assay, Recombinant, Purification, SDS Page

Journal: Molecular Cell

Article Title: Initiation of Quality Control during Poly(A) Translation Requires Site-Specific Ribosome Ubiquitination

doi: 10.1016/j.molcel.2016.11.039

Figure Lengend Snippet:

Article Snippet: GST-UBE1 (human) , Boston Biochem , Cat. #E-306.

Techniques: Recombinant, Protease Inhibitor, Methylation, Ubiquitin Proteomics, Expressing, Plasmid Preparation, Sequencing, Negative Control, Software

Journal: Developmental cell

Article Title: Ari-1 regulates myonuclear organization together with Parkin and is associated with aortic aneurysms

doi: 10.1016/j.devcel.2018.03.020

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: A 50 μL reaction mixture for KoiU2 ubiquitination contained 1μM human E1 recombinant protein (Enzo), 5μM UbcH7 (Boston Biochem), 2μM GST-tagged E3 ubiquitin ligase, 5μM T7-KoiU2, 5mM Mg-ATP (Enzo) 2.5μM biotinylated ubiquitin (Enzo) and 1mM DTT in 1× ubiquitination buffer (Enzo).

Techniques: Recombinant, Plasmid Preparation, Protease Inhibitor, Lysis, Transfection, SYBR Green Assay, Mutagenesis, Modification, shRNA