mouse uba1  (Addgene inc)

Journal: Molecular cell

Article Title: PEX5 translocation into and out of peroxisomes drives matrix protein import.

doi: 10.1016/j.molcel.2022.07.004

Figure Lengend Snippet: Figure 1. PEX5 recycling recapitulated in Xenopus egg extract (A) Xenopus eggs were lysed by centrifugation to generate an extract (the indicated layer between the lipid on top and cellular debris on bottom). The extract was treated with dimethylsulfoxide (DMSO) or the indicated E1 enzyme inhibitors dissolved in DMSO (UBA1, ubiquitin-activating enzyme; NAE, NEDD8-activating enzyme). Import activity was then assessed by incubating with the fluorescent cargo mScarlet-SKL and imaging on a spinning disk confocal microscope; repeated rounds of import into peroxisomes result in the appearance of bright puncta. The number of puncta in an imaged field was quantified relative to that in the untreated reaction (n = 9 fields per reaction; bars specify the median). (B) Upper scheme illustrates the strategy to deplete free ubiquitin from extract by inhibiting deubiquitinating enzymes (DUBs) with ubiquitin vinyl sulfone (UbVS); lower scheme shows the approach for restoring peroxisome import activity. On the right, extract was treated with buffer (mock) or UbVS and then supplemented either with buffer or wild-type ubiquitin, wild-type PEX5, or both components together. Import activity was then assessed as above (n = 16 fields per reaction). (legend continued on next page)

Article Snippet: The plasmid encoding mouse UBA1 was acquired from Addgene (no. 32534) and was described previously (Carvalho et al., 2012).

Techniques: Centrifugation, Ubiquitin Proteomics, Activity Assay, Imaging, Microscopy

Journal: Structure(London, England:1993)

Article Title: Structural Studies of HHARI/UbcH7∼Ub Reveal Unique E2∼Ub Conformational Restriction by RBR RING1

doi: 10.1016/j.str.2017.04.013

Figure Lengend Snippet:

Article Snippet: pET28 mouse Uba1 , , Addgene, Plasmid #32534.

Techniques: Virus, Recombinant, Clinical Proteomics, Ubiquitin Proteomics, Plasmid Preparation, Software

Journal: JCI Insight

Article Title: Systemic restoration of UBA1 ameliorates disease in spinal muscular atrophy

doi: 10.1172/jci.insight.87908

Figure Lengend Snippet: ( A – C ) Significant reduction of survival motor neuron (SMN) and ubiquitin-like modifier activating enzyme 1 (UBA1) protein in type I spinal muscular atrophy (SMA) patient iPSC-derived motor neurons, as quantified by Western blot (independent clones per genotype: control n = 9 and SMA n = 8; unpaired, 2-tailed Student’s t test). ( D – F ) Significant reduction in Smn and Uba1 protein levels in zebrafish injected with 6 ng morpholino oligonucleotide (MO) targeted against Smn compared to uninjected controls, as quantified Western blot analysis ( n = 3, batches of 30 fish per lane; unpaired, 2-tailed Student’s t test). Lanes were run on the same gel but were noncontiguous. ( G ) Representative micrographs of spinal motor axons from uninjected control zebrafish, zebrafish injected with 4 ng Smn MO (white arrow indicates abnormal axon branching, gray arrow indicates severely truncated axons), and zebrafish injected with 4 ng Smn MO coinjected 200 ng/μl human UBA1 mRNA at 30 hours after fertilization (scale bar: 50 μm). ( H – J ) Significant improvement in the percentage of normal, branched, and severely truncated motor axons in zebrafish injected with 4 ng Smn MO coinjected 200 ng/μl human UBA1 mRNA ( n = 20 per treatment group; 1-way ANOVA with Tukey’s post-hoc test). ( K ) Representative tracings of automated swim path analysis of uninjected control zebrafish, zebrafish injected with 4 ng Smn MO, and zebrafish injected with 4 ng Smn MO coinjected 200 ng/μl human UBA1 mRNA at 3 days after fertilization (scale bar: 1 cm). ( L ) Significant improvement in the number of quadrants entered during automated swim path analysis of zebrafish injected with 4 ng Smn MO coinjected 200 ng/μl human UBA1 mRNA (control n = 18, Smn MO n = 33, Smn MO + UBA1 n = 32; Kruskal-Wallis test with Dunn’s post-hoc test). * P ≤ 0.05, ** P ≤ 0.01, **** P ≤ 0.001.

Article Snippet: Sections were permeabilized in 0.3% Triton X-100 in PBS and then blocking solution (4% BSA, 0.3% Triton X-100 in PBS) for 30 minutes at room temperature before overnight incubation with primary antibody solution at 4°C: rabbit anti-GFP (1:500, Abcam, ab290), mouse anti-UBA1 (1:200, Sigma-Aldrich, E3125), and rat anti-Ly76 (1:100, Abcam, ab91113).

Techniques: Derivative Assay, Western Blot, Clone Assay, Injection

Journal: JCI Insight

Article Title: Systemic restoration of UBA1 ameliorates disease in spinal muscular atrophy

doi: 10.1172/jci.insight.87908

Figure Lengend Snippet: ( A and B ) Adeno-associated virus serotype 9–ubiquitin-like modifier activating enzyme 1 (AAV9-UBA1) delivered at P1 significantly increased UBA1 protein levels in spinal cord, gastrocnemius muscle, heart, liver, lung, and kidney in P7 control mice ( n = 3 mice per group; unpaired 2-tailed Student’s t test). Lanes were run on the same gel but were noncontiguous. ( C ) PCR products following mouse, human, and UBA1 viral cDNA amplification using primers detecting both mouse Uba1 and human UBA1 cDNA (top row), unique to mouse Uba1 cDNA (middle row), and unique to human UBA1 cDNA (bottom row). ( D ) Significant increase in AAV-expressed human UBA1 mRNA, but not endogenous mouse Uba1 mRNA, in the hearts of P7 AAV9-UBA1–treated mice ( n = 3 mice per group; unpaired, 2-tailed Student’s t test). ( E ) Representative Nissl-stained spinal cords (ventral horn) from uninjected control and AAV9-UBA1–treated control mice at P9 (scale bar: 250 μm). ( F ) No significant change in the number of motor neurons in the spinal cords of AAV9-UBA1–treated control P9 mice ( n = 4 mice per group; unpaired, 2-tailed Student’s t test). ( G ) Representative confocal micrographs of neuromuscular junctions (NMJs) in the external oblique from uninjected control and AAV9-UBA1–treated control mice at P9 (axonal inputs in green; postsynaptic endplates in red; scale bar: 25 μm). ( H ) No significant change in axonal inputs at the NMJ in the external oblique muscle of AAV9-UBA1–treated control mice at P9 ( n = 4 mice per group; unpaired, 2-tailed Student’s t test). ( I ) Uninjected control and AAV9-UBA1–treated control mice at P180 (scale bar: 2 cm). ( J – L ) No change in AAV9-UBA1–treated controls from P1 to P180 with respect to ( J ) weight (at P180 n = 4 mice per group), ( K) survival (Kaplan-Meier survival analysis), or ( L ) righting reflex test performance (from P1 to P12–P14; unpaired, 2-tailed Student’s t test at each time point). ns (not significant) P > 0.05 , * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.005, **** P ≤ 0.001. As part of a different comparative analysis, uninjected littermate control data concerning motor neuron cell body counts ( F ) and NMJ axonal input ( H ) is also shown in , respectively. Weight, survival and righting times ( J – L ) for uninjected controls are also shown in .

Article Snippet: Sections were permeabilized in 0.3% Triton X-100 in PBS and then blocking solution (4% BSA, 0.3% Triton X-100 in PBS) for 30 minutes at room temperature before overnight incubation with primary antibody solution at 4°C: rabbit anti-GFP (1:500, Abcam, ab290), mouse anti-UBA1 (1:200, Sigma-Aldrich, E3125), and rat anti-Ly76 (1:100, Abcam, ab91113).

Techniques: Amplification, Staining

Journal: JCI Insight

Article Title: Systemic restoration of UBA1 ameliorates disease in spinal muscular atrophy

doi: 10.1172/jci.insight.87908

Figure Lengend Snippet: ( A ) Quantification of ubiquitin-like modifier activating enzyme 1 (Uba1) levels in the spinal cord, gastrocnemius muscle, heart, liver, lung, kidney, and brain of spinal muscular atrophy (SMA) mice at P1, P3, P7, and P11 by western blot analysis, expressed as a percentage of control values (n = 3 mice for each genotype at each time point). ( B and C ) Intravenous adeno-associated virus serotype 9–UBA1 (AAV9-UBA1) gene therapy at P1 leads to a significant increase in UBA1 protein levels in the spinal cord, gastrocnemius muscle, heart, liver, lung, and kidney (but not whole brain) in P7 SMA mice, as quantified by Western blot ( n = 3 mice per treatment group, except for spinal cord, for which n = 5; unpaired 2-tailed Student’s t test). Lanes were run on the same gel but were noncontiguous. ( D ) Representative confocal micrographs showing increased UBA1 levels (green) in the heart, liver, and motor neurons in the spinal cord ventral horn of P7 AAV9-UBA1–treated mice compared to uninjected SMA mice. Hearts and livers were colabeled with DAPI and the spinal cord fluorescent Nissl stain (blue) (scale bar: 50 μm). ns (not significant) P > 0.05 , * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.005.

Article Snippet: Sections were permeabilized in 0.3% Triton X-100 in PBS and then blocking solution (4% BSA, 0.3% Triton X-100 in PBS) for 30 minutes at room temperature before overnight incubation with primary antibody solution at 4°C: rabbit anti-GFP (1:500, Abcam, ab290), mouse anti-UBA1 (1:200, Sigma-Aldrich, E3125), and rat anti-Ly76 (1:100, Abcam, ab91113).

Techniques: Western Blot, Staining

Journal: JCI Insight

Article Title: Systemic restoration of UBA1 ameliorates disease in spinal muscular atrophy

doi: 10.1172/jci.insight.87908

Figure Lengend Snippet: ( A ) Average weights of adeno-associated virus serotype 9–ubiquitin-like modifier activating enzyme 1–treated (AAV9-UBA1–treated) control mice ( n = 28 at P3), uninjected spinal muscular atrophy (SMA) mice ( n = 25 at P3), and AAV9-UBA1–treated SMA mice ( n = 19 at P3). ( B ) Survival analysis of AAV9-UBA1–treated control mice ( n = 19), uninjected SMA mice ( n = 25), and AAV9-UBA1–treated SMA mice ( n = 11) (Kaplan-Meier survival analysis). ( C ) Average righting reflex times of AAV9-UBA1–treated control mice ( n = 28 at P3), uninjected SMA mice ( n = 25 at P3), and AAV9-UBA1–treated SMA mice ( n = 19 at P3). ( D ) Representative photographs of a AAV9-UBA1–treated control mouse, uninjected SMA mouse, and a AAV9-UBA1–treated SMA mouse at P9 (scale bar: 2 cm). **** P ≤ 0.001. As part of a different comparative analysis, weight, survival and righting times ( A – C ) for uninjected controls are also shown in .

Article Snippet: Sections were permeabilized in 0.3% Triton X-100 in PBS and then blocking solution (4% BSA, 0.3% Triton X-100 in PBS) for 30 minutes at room temperature before overnight incubation with primary antibody solution at 4°C: rabbit anti-GFP (1:500, Abcam, ab290), mouse anti-UBA1 (1:200, Sigma-Aldrich, E3125), and rat anti-Ly76 (1:100, Abcam, ab91113).

Techniques:

Journal: JCI Insight

Article Title: Systemic restoration of UBA1 ameliorates disease in spinal muscular atrophy

doi: 10.1172/jci.insight.87908

Figure Lengend Snippet: ( A ) Nissl-stained spinal cords (ventral horn) from uninjected control, uninjected spinal muscular atrophy (SMA), and adeno-associated virus serotype 9–ubiquitin-like modifier activating enzyme 1–treated (AAV9-UBA1–treated) SMA mice at P9 (scale bar: 250 μm). ( B ) Significant rescue of motor neurons in AAV9-UBA1–treated SMA mice at P9 ( n = 4 mice per group). ( C ) Confocal micrographs of neuromuscular junctions (NMJs) in the external oblique from uninjected control, uninjected SMA, and AAV9-UBA1–treated SMA mice at P9 (axonal inputs in green; postsynaptic endplates in red; scale bar: 25 μm). ( D ) Significant improvement in NMJ pathology in the external oblique muscle of AAV9-UBA1–treated SMA mice at P9 ( n = 4 mice per group). ( E ) Bright-field micrographs of individual teased muscle fibers from the external oblique of uninjected control, uninjected SMA, and AAV9-UBA1–treated SMA mice at P9 (scale bar: 100 μm). ( F ) Significant rescue of muscle fiber diameters in AAV9-UBA1–treated SMA mice at P9 ( n = 4 mice per group). One-way ANOVA with Tukey’s post-hoc test for all analyses. * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.005, **** P ≤ 0.001. As part of a different comparative analysis, uninjected littermate control data motor neuron cell body counts ( B ) and NMJ axonal input ( D ) is also shown in , respectively.

Article Snippet: Sections were permeabilized in 0.3% Triton X-100 in PBS and then blocking solution (4% BSA, 0.3% Triton X-100 in PBS) for 30 minutes at room temperature before overnight incubation with primary antibody solution at 4°C: rabbit anti-GFP (1:500, Abcam, ab290), mouse anti-UBA1 (1:200, Sigma-Aldrich, E3125), and rat anti-Ly76 (1:100, Abcam, ab91113).

Techniques: Staining

Journal: JCI Insight

Article Title: Systemic restoration of UBA1 ameliorates disease in spinal muscular atrophy

doi: 10.1172/jci.insight.87908

Figure Lengend Snippet: ( A ) Uninjected control, uninjected spinal muscular atrophy (SMA), and adeno-associated virus serotype 9–ubiquitin-like modifier activating enzyme 1–treated (AAV9-UBA1–treated) SMA mouse hearts and livers at P9 (scale bar: 0.5 cm). ( B – D ) Significant improvement in heart pathology in P9 AAV9-UBA1–treated SMA mice for the parameters of ( B ) heart length ( n = 4 mice per group), ( C ) heart weight (control n = 5, SMA n = 11, SMA + AAV9-UBA1 n = 4), and ( D ) heart-weight-to-body-weight ratio (control n = 5, SMA n = 7, SMA + AAV9-UBA1 n = 4). ( E ) Histological analysis of uninjected control, uninjected SMA, and AAV9-UBA1 SMA P9 livers. Top row shows H&E-stained micrographs (white arrows indicate hematopoietic islands; black arrows indicate megakaryocytes; scale bar: 25 μm). Middle row shows micrographs of Ly76 immunohistochemistry (scale bar: 25 μm). Bottom row shows micrographs (scale bar: 25 μm) along their magnified insets (scale bar: 12.5 μm) costained with the Ly76 marker (green) and DAPI (blue). ( F and G ) Significant improvements in ( F ) liver Ly76 + erythrocyte area and ( G ) liver erythrocyte precursor cell area of AAV9-UBA1–treated SMA mice at P9 ( n = 4 mice per group). One-way ANOVA with Tukey’s post-hoc test for all analyses. ** P ≤ 0.01, *** P ≤ 0.005, **** P ≤ 0.001.

Article Snippet: Sections were permeabilized in 0.3% Triton X-100 in PBS and then blocking solution (4% BSA, 0.3% Triton X-100 in PBS) for 30 minutes at room temperature before overnight incubation with primary antibody solution at 4°C: rabbit anti-GFP (1:500, Abcam, ab290), mouse anti-UBA1 (1:200, Sigma-Aldrich, E3125), and rat anti-Ly76 (1:100, Abcam, ab91113).

Techniques: Staining, Immunohistochemistry, Marker

Journal: JCI Insight

Article Title: Systemic restoration of UBA1 ameliorates disease in spinal muscular atrophy

doi: 10.1172/jci.insight.87908

Figure Lengend Snippet: ( A – D ) Western blot analysis of β-catenin, polyubiquitin, and monoubiquitin protein levels in uninjected spinal muscular atrophy (SMA) and adeno-associated virus serotype 9–ubiquitin-like modifier activating enzyme 1 (AAV9-UBA1) SMA mouse hearts at P7 ( n = 3 mice per treatment group; unpaired 2-tailed Student’s t test). Lanes were run on the same gel but were noncontiguous. ( E – G ) Western blot analysis of UBA1 and survival motor neuron (SMN) protein levels in uninjected control, uninjected SMA, AAV9-UBA1–treated control, and AAV9-UBA1–treated SMA P7 hearts ( n = 3 mice per group; 1-way ANOVA with Tukey’s post-hoc test). ( H ) PCR products following mouse, human, and UBA1 viral cDNA amplification using primers that detect full-length SMN ( FL-SMN ) (top row) or Δ 7-SMN (bottom row). ( I ) Significant increase in FL-SMN , but not Δ 7-SMN , mRNA expression in the hearts of AAV9-UBA1–treated mice at P7, as detected by qRT-PCR quantification using the primers shown in H ( n = 3 mice per treatment group; unpaired 2-tailed Student’s t test). ns (not significant) P > 0.05 , * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.005, **** P ≤ 0.001.

Article Snippet: Sections were permeabilized in 0.3% Triton X-100 in PBS and then blocking solution (4% BSA, 0.3% Triton X-100 in PBS) for 30 minutes at room temperature before overnight incubation with primary antibody solution at 4°C: rabbit anti-GFP (1:500, Abcam, ab290), mouse anti-UBA1 (1:200, Sigma-Aldrich, E3125), and rat anti-Ly76 (1:100, Abcam, ab91113).

Techniques: Western Blot, Amplification, Expressing, Quantitative RT-PCR