e1 uba1 addgene clone 34965  (Addgene inc)

Journal: PLoS ONE

Article Title: Characterization of Ubiquitin-Activating Enzyme Uba1 in the Nucleus by Its Mammalian Temperature-Sensitive Mutant

doi: 10.1371/journal.pone.0096666

Figure Lengend Snippet: (A) Sequencing traces obtained with Uba1 DNA (prepared by reverse transcription of pools of mRNA or from genomic DNA) from wild-type CHO-K1 and mutant tsTM3 cells. The wild type contains a G at nucleotide 768 of Uba1 , whereas the mutant contains an A. The G-to-A transition at nucleotide 768 converts Met to Ile at amino acid 256. (B) Structure of mammalian Uba1. Colored boxes represent four functional domains, adenylation domains (IAD and AAD), catalytic cysteine half-domains (FCCH and SCCH), four-helix bundle domain (4HB), and C-terminal ubiquitin-fold domain (UFD). Locations of the mutation found in ts mutant ts20 and in X-linked spinal muscular atrophy (XL-SMA) are shown above the diagram. Alignments were made with CLUSTAL W relative to positions 203–301 of hamster Uba1 (Accession No. AB661372) with sequences from Homo sapiens (H. s.; NCBI Gene ID: 7317), Mus musculus (M. m.; ID: 22201), and Rattus norvegicus (R. n.; ID: 314432). Asterisks indicate identical residues.

Article Snippet: The Bgl II- Sal I restriction fragment containing the coding sequence of hamster Uba1 cDNA was cloned into the vectors pEGFP-C1 and pEGFP-N3 (Clontech Laboratories, Palo Alto, CA) to yield the plasmids GFP-Uba1 and Uba1-GFP, respectively.

Techniques: Sequencing, Mutagenesis, Functional Assay

Journal: PLoS ONE

Article Title: Characterization of Ubiquitin-Activating Enzyme Uba1 in the Nucleus by Its Mammalian Temperature-Sensitive Mutant

doi: 10.1371/journal.pone.0096666

Figure Lengend Snippet: (A) Western blot analysis of Uba1 in tsTM3 cells. Wild-type CHO-K1 or ts mutant tsTM3 cells were grown at 34°C or incubated at 39°C for the times shown in the figure and lysed. The proteins resolved on acrylamide gels and were detected by immunoblotting with an antibody directed against Uba1 (rabbit polyclonal antibody supplied from Rockland). Uba1 exists as two isoforms: Uba1A (∼117 kDa), localized predominantly in the nucleus and Uba1B (∼110 kDa), localized in the cytoplasm. Similar results were obtained from another antibody (rabbit polyclonal from Calbiochem). Only relevant parts of the blot are shown. SMC3 was included as a loading control. Temperature-dependent reduction in the amount of Uba1 was found in tsTM3 cells. (B, C) Quantitative analyses of the amounts of Uba1 in CHO-K1 and tsTM3 cells. Band intensities, like those shown in panel (A), were measured and expressed relative to that of each at 34°C or relative to the lower band at 34°C with standard deviation of at least three experiments. Bands of Uba1B at 34°C in each cell are selected as a control to compare the amount of Uba1 because it appeared to be permanent. P values were calculated by Student t -test. Significant differences from values at 34°C (B, gray bars; C, solid gray bars) are shown by asterisks. Incubation at 39°C resulted in a significant decrease in Uba1A in tsTM3 cells, although the proportion of Uba1A to Uba1B in mutant cells at 34°C was smaller than that of wild-type CHO-K1.

Article Snippet: The Bgl II- Sal I restriction fragment containing the coding sequence of hamster Uba1 cDNA was cloned into the vectors pEGFP-C1 and pEGFP-N3 (Clontech Laboratories, Palo Alto, CA) to yield the plasmids GFP-Uba1 and Uba1-GFP, respectively.

Techniques: Western Blot, Mutagenesis, Incubation, Standard Deviation

Journal: PLoS ONE

Article Title: Characterization of Ubiquitin-Activating Enzyme Uba1 in the Nucleus by Its Mammalian Temperature-Sensitive Mutant

doi: 10.1371/journal.pone.0096666

Figure Lengend Snippet: (A) Isoform of Uba1 tagged with GFP and its localization. Cell lines expressing GFP-Uba1 or Uba1-GFP constructs were isolated from the ts mutant cell line, tsTM3. Uba1 tagged with GFP complements deficiencies in tsTM3 cells and allows them to grow normally at 39°C. Cells were counterstained with Hoechst 33342. The fluorescent Uba1 in the GFP-Uba1 derivative is mainly nuclear, whereas the other derivative, Uba1-GFP, contains higher concentrations in both nucleus and cytoplasm. Bar, 10 µm. (B) Western blot analysis of Uba1 tagged with GFP. Cells expressing GFP-Uba1 or Uba1-GFP, as well as parental (tsTM3) and grandparental (CHO-K1) cells were lysed and the proteins resolved on acrylamide gels. Six samples of each distinct GFP clone were analyzed: lanes 3–8, GFP-Uba1; lanes 9–14, Uba1-GFP. Different forms of Uba1 were detected by immunoblotting with antibodies directed against Uba1 and GFP. Only relevant parts of the blot are shown. α-tubulin was included as a loading control. Positions of endogenous and hybrid Uba1 are shown. Band intensities relative to that of the endogenous cytoplasmic form of Uba1 in each of the clones are presented below the blot of Uba1. A derivative of tsTM3 cells, tm3UG16, appears to express very little Uba1-GFP, suggesting the possibility of spontaneous reversion. It is also possible that the rescue depends on a cleaved form of hybrid that resembles the endogenous form but which can no longer be detected via fluorescence. Slightly different migration of bands is observed in tm3UG11. (C) Images of living tsTM3 cells expressing Uba1 tagged with GFP. Cells were counterstained with Hoechst 33342. Upper and middle rows represent living interphase cells expressing the GFP-Uba1 construct. The fluorescent Uba1 in a derivative, tm3GU1, is predominantly in the nucleus. Living mitotic cells (bottom). Bar, 10 µm.

Article Snippet: The Bgl II- Sal I restriction fragment containing the coding sequence of hamster Uba1 cDNA was cloned into the vectors pEGFP-C1 and pEGFP-N3 (Clontech Laboratories, Palo Alto, CA) to yield the plasmids GFP-Uba1 and Uba1-GFP, respectively.

Techniques: Expressing, Construct, Isolation, Mutagenesis, Western Blot, Clone Assay, Fluorescence, Migration

Journal: PLoS ONE

Article Title: Characterization of Ubiquitin-Activating Enzyme Uba1 in the Nucleus by Its Mammalian Temperature-Sensitive Mutant

doi: 10.1371/journal.pone.0096666

Figure Lengend Snippet: (A) Diagram of the structures of the full-length and truncated forms of Uba1. Boxes represent the domains of Uba1 in . (B) Photographs of colonies of tsTM3 cells after transfection of 2 µg of plasmid DNAs encoding GFP hybrids of Uba1 or Uba1D1-40 and vectors. After 14 days of incubation at 39°C or 34°C with 400 µg/ml G418, the colonies on the dishes were stained with methylene blue. Cells survived at 39°C after transfection of Uba1 derivatives; however, relatively few cells and no cells grew after transfection of Uba1D1-40 derivatives and vectors, respectively. There was no difference in the colony formation between plasmid DNAs encoding GFP hybrids of Uba1 and Uba1D1-40 after the selection for resistance to G418. (C) Quantitative analysis of colony formation at 39°C. Colonies such as those shown in panel (B) were counted. The colony formation efficiencies of 39°C were normalized to those of 34°C with G418 and are expressed with a standard deviation of at least three experiments. P values were calculated by Student t -test. Significant differences from the efficiency of GFP-Uba1 are shown by asterisks.

Article Snippet: The Bgl II- Sal I restriction fragment containing the coding sequence of hamster Uba1 cDNA was cloned into the vectors pEGFP-C1 and pEGFP-N3 (Clontech Laboratories, Palo Alto, CA) to yield the plasmids GFP-Uba1 and Uba1-GFP, respectively.

Techniques: Transfection, Plasmid Preparation, Incubation, Staining, Selection, Standard Deviation

Journal: PLoS ONE

Article Title: Characterization of Ubiquitin-Activating Enzyme Uba1 in the Nucleus by Its Mammalian Temperature-Sensitive Mutant

doi: 10.1371/journal.pone.0096666

Figure Lengend Snippet: (A) Western blot analysis of Fucci in CHO-K1 or tsTM3 cells. Wild-type CHO-K1 or ts mutant tsTM3 cells expressing Fucci-G 1 Orange or Fucci-S/G 2 /M Green were grown at 34°C or incubated at 39°C for the times shown in the figure and lysed. The proteins resolved on acrylamide gels and were detected by immunoblotting with antibodies directed against fluorescent tags (mKO2 or mAG1) and Uba1. Only relevant parts of the blot are shown. α-tubulin is included as a loading control. Temperature-dependent increases in the amount of Fucci with the reduction of Uba1 were found in tsTM3 cells. (B) Quantitative analysis of bands in blots of tsTM3 cells expressing Fucci. Band intensities of Fucci and Uba1, like those shown in panel (A), were measured and are expressed relative to that of each at 34°C or relative to the lower band at 34°C with standard deviation of at least three experiments. P values were calculated by Student t -test. Significant differences from values at 34°C are shown by asterisks. Incubation at 39°C resulted in a significant increase of Fucci (mKO2-hCdt1 or mAG1-hGem) with decrease in Uba1A in tsTM3 cells.

Article Snippet: The Bgl II- Sal I restriction fragment containing the coding sequence of hamster Uba1 cDNA was cloned into the vectors pEGFP-C1 and pEGFP-N3 (Clontech Laboratories, Palo Alto, CA) to yield the plasmids GFP-Uba1 and Uba1-GFP, respectively.

Techniques: Western Blot, Mutagenesis, Expressing, Incubation, Standard Deviation

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: iScience

Article Title: A series of xanthenes inhibiting Rad6 function and Rad6-Rad18 interaction in the PCNA ubiquitination cascade

doi: 10.1016/j.isci.2022.104053

Figure Lengend Snippet: Half-maximal inhibitory concentration (IC 50 ) values by Alpha assays against PCNA ubiquitination, Uba1∼ubiquitin thioester formation, Rad6∼ubiquitin thioester formation, Rad18 autoubiquitination, Rad6–Rad18 interaction, and Mms2–Ubc13∼ubiquitin thioester formation

Article Snippet: His-tagged human Uba1 , Addgene , Cat#63571, BIL3039 (lab code).

Techniques: Concentration Assay, Ubiquitin Proteomics, Inhibition

Journal: iScience

Article Title: A series of xanthenes inhibiting Rad6 function and Rad6-Rad18 interaction in the PCNA ubiquitination cascade

doi: 10.1016/j.isci.2022.104053

Figure Lengend Snippet: Dose response for Uba1∼ubiquitin thioester formation in the presence of compounds by Alpha assay (A) Dose response of NSC 157411 for Uba1~ubiquitin thioester formation. (B) Dose response of NSC 119888 for Uba1~ubiquitin thioester formation. Curves in (A) and (B) were fitted by nonlinear regression and graphed semilogarithmically. (C) Bar graph representation of inactive compounds. Data represent mean with SD for triplicate samples in each case.

Article Snippet: His-tagged human Uba1 , Addgene , Cat#63571, BIL3039 (lab code).

Techniques: Ubiquitin Proteomics

Journal: iScience

Article Title: A series of xanthenes inhibiting Rad6 function and Rad6-Rad18 interaction in the PCNA ubiquitination cascade

doi: 10.1016/j.isci.2022.104053

Figure Lengend Snippet:

Article Snippet: His-tagged human Uba1 , Addgene , Cat#63571, BIL3039 (lab code).

Techniques: Virus, Recombinant, Ubiquitin Proteomics, Modification, Amplified Luminescent Proximity Homogenous Assay, Plasmid Preparation, Software