signal seeker ubiquitin detection kit  (Cytoskeleton Inc)

Journal: Nature Communications

Article Title: Mitochondrial damage triggers the concerted degradation of negative regulators of neuronal autophagy

doi: 10.1038/s41467-025-62379-5

Figure Lengend Snippet: A – D Proteasomal inhibition blocks the concerted degradation of MTMR5, MTMR2, and Rubicon. A Representative western blots from lysates of WT murine embryonic cortical neurons treated with vehicle (EtOH) or 10 μM MG132 for 1 hr followed by an additional treatment with vehicle (EtOH) or 15 nM Ant A for 2 hrs. B MTMR5 band intensity normalized to total protein from WT neurons treated with EtOH/MG132 and EtOH/Ant A. C Ratio of intact MTMR2 normalized to total (intact + degraded) band intensities from WT neurons treated with EtOH/MG132 and EtOH/Ant A. D Rubicon band intensity normalized to total protein from WT neurons treated with EtOH/MG132 and EtOH/Ant A ( A – D : N = 3 experiments, one way ANOVA with Sidak’s multiple comparison test). E , F Mitochondrial damage accelerates ubiquitination of Rubicon in neurons. E Representative western blot from wild type cortical neuronal lysates treated with vehicle (EtOH) or 30 nM Ant A for 2 hrs and immunoprecipitated with Rubicon antibody or control Rabbit IgG. F Quantification of the ratio of ubiquitinated to total Rubicon upon immunoprecipitation of Rubicon in neurons treated with EtOH or Ant A ( N = 4 experiments, two-tailed unpaired t test). G – K Negative regulators of autophagy are ubiquitinated in response to mitochondrial stress. G Representative western blot of ubiquitin enrichment assay from lysates of WT murine embryonic cortical neurons. Neurons were treated with 10 μM MG132 for 1 hr followed by an additional treatment with 15 nM Ant A for another 2 hrs (Ub beads = Ubiquitination affinity beads). H Ub enrichment following pull-down with Ub beads or control beads, normalized to intensity in the input lane. I MTMR5 enrichment following pull-down with Ub beads or control beads, normalized to intensity in the input lane. J MTMR2 enrichment following pull-down with Ub beads or control beads, normalized to its intensity in the input lane. K Rubicon enrichment after pull-down with Ub beads or control beads, normalized to its intensity in the input lane ( G – K : N = 3 experiments, two-tailed unpaired t test). Error bars indicate SEM. Source data are provided as a Source Data file.

Article Snippet: The assay was performed using the Ubiquitin Detection Kit (Cytoskeleton, BK161), following the steps as mentioned in the manufacturer’s protocol, with some modifications.

Techniques: Inhibition, Western Blot, Comparison, Ubiquitin Proteomics, Immunoprecipitation, Control, Two Tailed Test