Journal: EMBO reports

Article Title: UBXD8 mediates mitochondria-associated degradation to restrain apoptosis and mitophagy.

doi: 10.15252/embr.202254859

Figure Lengend Snippet: Figure 4. UBXD8 degrades mitochondrial and endoplasmic reticulum (ER) substrates in cis and in trans.

Article Snippet: We obtained a monoclonal UBXD8 antibody (#34945, CST) for immunofluorescence staining.

Techniques:

Journal: Cell reports

Article Title: Interactomic analysis reveals a homeostatic role for the HIV restriction factor TRIM5α in mitophagy

doi: 10.1016/j.celrep.2022.110797

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Anti-ETEA Antibody (F-7) , Santa Cruz Biotechnology , sc-374098; RRID: AB_10918565.

Techniques: Ubiquitin Proteomics, Purification, Virus, Recombinant, Protease Inhibitor, Magnetic Beads, Lysis, Western Blot, Stripping, Staining, Transfection, Luciferase, Isolation, In Situ, Mutagenesis, Knock-Out, Stable Transfection, Expressing, Software, Imaging

Journal: Cell reports

Article Title: Interactomic analysis reveals a homeostatic role for the HIV restriction factor TRIM5α in mitophagy

doi: 10.1016/j.celrep.2022.110797

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Anti-ETEA Antibody (F-7) , Santa Cruz Biotechnology , sc-374098; RRID: AB_10918565.

Techniques: Ubiquitin Proteomics, Purification, Virus, Recombinant, Protease Inhibitor, Magnetic Beads, Lysis, Western Blot, Stripping, Staining, Transfection, Luciferase, Isolation, In Situ, Mutagenesis, Knock-Out, Stable Transfection, Expressing, Software, Imaging

Journal: Journal of medicinal chemistry

Article Title: Activation of E6AP/UBE3A-Mediated Protein Ubiquitination and Degradation Pathways by a Cyclic γ -AA Peptide

doi: 10.1021/acs.jmedchem.1c01922

Figure Lengend Snippet: Effect of the γ-AA peptide ligands on the UB ligase activity of E6AP assayed by substrate ubiquitination. (a) Measuring the effects of ligands P1–P8 on UbxD8 ubiquitination catalyzed by E6AP. The ubiquitination reaction contains ligands (10 or 100 μM) incubating with the Uba1-UbcH7-E6AP cascade and UbxD8 as the E6AP substrate. Ligand P6 showed the most significant stimulatory effect on UbxD8 ubiquitination catalyzed by E6AP. (b) Dose-dependent activation of E6AP-catalyzed substrate ubiquitination by the P6 ligand. Varying concentrations of P6 were added to the ubiquitination reaction containing the Uba1-UbcH7-E6AP cascade and the E6AP substrates UbxD8 (left panel), HHR23A (middle panel), and β-catenin (right panel). Ubiquitination of the substrates was measured by Western blots of the reaction probed with antibodies specific for each substrate. (c) Ligands P1–P8 have no stimulatory effect on p53 ubiquitination catalyzed by E6AP pairing with the E6 protein of HPV. The ligands (10 or 100 μM) were incubated with the E6AP enzymatic cascade, the viral E6 protein, and p53. The ubiquitination of p53 was followed by a Western blot of the reaction mixture probed with an anti-p53 antibody.

Article Snippet: The anti-HHR23A antibody (sc-365669), anti-UB antibody (sc-8017), anti-UbxD8 antibody (sc-374098), anti-E6AP antibody (sc-25509), anti- β -actin (sc-47778), anti- β -catenin antibody(sc-65480), and anti-p53 antibody (sc-126) were from Santa Cruz Biotechnology.

Techniques: Activity Assay, Activation Assay, Western Blot, Incubation

Journal: Journal of medicinal chemistry

Article Title: Activation of E6AP/UBE3A-Mediated Protein Ubiquitination and Degradation Pathways by a Cyclic γ -AA Peptide

doi: 10.1021/acs.jmedchem.1c01922

Figure Lengend Snippet: Stimulatory effect of the P6 ligand on E6AP-catalyzed substrate ubiquitin in the cell and acceleration of the degradation of E6AP substrates. (a) P6 enhanced the ubiquitination of E6AP substrates UbxD8 (left panel) and HHR23A (right panel) in HEK293T cells. The P6 peptide of 0, 25, and 50 μM was incubated with HEK293T cells for 14 h, and the cells were treated with proteasome inhibitor MG132 for another 12 h. The cells were harvested for immunoprecipitating the substrate proteins with specific antibodies from the cell lysate, and the ubiquitination levels of the substrate proteins were measured by Western blots probed with an anti-UB antibody. *, IgG light chain (LC); **, IgG heavy chain (HC). (b) Cycloheximide (CHX) chase assay to measure the accelerated degradation of UbxD8 (left panels) and HHR23A (middle panels) in the presence of varying concentrations of P6. β-actin levels in the cell were measured in parallel as a control (right panels). Cells were incubated with varying concentrations of the P6 peptide for 14 h and treated with ribosome inhibitor CHX for 0, 1, and 3 h before harvesting to collect cell lysates. Levels of UbxD8, HHR23A, and β-actin in the cell lysates were measured by Western blot probed with specific antibodies. (c) Levels of the E6AP substrates UbxD8 (top panel) and HHR23A (bottom panel) were plotted against the chase time after the cells were incubated with varying concentrations of the P6 peptide. P6 accelerated UbxD8 degradation at a concentration of 5 μM and accelerated HHR23A degradation at a concentration of 1 μM. Error bars = standard deviation from three independent experiments.

Article Snippet: The anti-HHR23A antibody (sc-365669), anti-UB antibody (sc-8017), anti-UbxD8 antibody (sc-374098), anti-E6AP antibody (sc-25509), anti- β -actin (sc-47778), anti- β -catenin antibody(sc-65480), and anti-p53 antibody (sc-126) were from Santa Cruz Biotechnology.

Techniques: Incubation, Western Blot, Concentration Assay, Standard Deviation