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This indicates that the nuclear export of SUMO2V1 is substantially increased upon cold-shock in HEK293A cells. 3. do not have labile H-atom. Three of the cell types analyzed were well-characterized cell lines exhibiting hypotriploid chromosomal numbers, thus PBMCs were included in our analyses to provide some degree of comparison with a population of normal cells. Q: What is the major organic product obtained from the following sequence of reactions? Each fraction was subsequently mixed with 200 μL of 100% ethanol, and the resulting mixes were transferred into a spin column, and centrifuged for 1 min at 3500×g. Emerging roles of sumoylation in the regulation of actin, microtubules, intermediate filaments, and septins. The PVDF membranes were blocked in 1 × Blocking Solution (1 × PBS + 3% fat-free milk + 0. Q: 4 Predict the product of the following reaction.

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Development of plasmid constructs coding for His-S-tagged SUMO2, the His-S-tagged SUMO alphas, and the His-S-YFP-tagged SUMOs and SUMO alphas. Q: What is the major elimination product obtained from an E2 reaction of each of the following alkyl…. Related Chemistry Q&A. MG132 treatment also increased the signal of all SUMOs, thus supporting proteasomal degradation as part of the regulatory mechanisms that control SUMO levels in the cell (data not shown). Xo's CH3 1) Hg(CH3COO)₂/H₂0 2) NaBH4 D. A: -> Hg(CH3COO)2/H2O, NaBH4 is reagent used for oxymercuration - demercurstion. Considering that SUMOylation is now recognized as a mediator of some of the liquid–liquid phase separation events that result in the formation of membrane-less organelles 60, it is possible that the non-conjugatable SUMO alphas may lack the ability to drive liquid–liquid phase separation events, thus explaining their decreased association to speckles and increased diffuse distribution.

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If the sequence match was longer than the length of the query, the additional nucleotides had to match the extended sequence of the query (that is, including additional 5' and 3' sequences that surround the one used as query). Note: The main thing to note while solving conversion reactions is to be thorough with named reactions and the reagents used for basic conversions. To this end, we compared the predominant cellular localization of the SUMO alphas with that of their respective prototypical SUMO proteins. Questions from AMU 2010. In preparation for development, membranes were washed 3 times with 1 × TPBS and 1 time with 1 × PBS. Having confirmed that the SUMO alphas are translated in human cells, we aimed to assess the functional properties of the SUMO alphas. Human embryonic kidney cells (HEK293A) were from Invitrogen (ThermoFisher Scientific, Inc., Waltham, MA). The nucleo-cytoplasmic distribution of the SUMO variants is differentially affected by cold-shock. Nuclear vs cytosolic fractionation. Our data indicate that all the variants coding for the SUMO alpha isoforms are exported to the cytoplasm, albeit with different efficiencies, and are actively translated by ribosomes, as supported by the finding of sequences specific for such variants among the pools of Ribo-seq data analyzed. Aliquots of the PCR products obtained were also analyzed by agarose gel electrophoresis using 1. Carlos Ontiveros and Alejandra Flores received support from the MARC program. Q: Question attached. Both analyses predicted that SUMO1α and SUMO2α contained substantial alterations in the characteristic β-grasp fold structure of their prototypical isoforms.

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To produce the SUMO3α coding construct, primers were designed to amplify the full-length of the pcDNA5/FRT/TO/His-S-SUMO3/IRES/HA-Ubc9 plasmid and produce a linear product with ends located around the region where the additional sequence is introduced by alternative splicing of the transcript. 1) A diethyl ether 2) H30* PB13 Mg…. The mechanisms responsible for the global increases in cellular SUMOylation triggered by stress remain to be fully characterized. 4) High-resolution melting curve with an initial stage of 60 °C for 1 min, a ramp of 0.

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A: We are having Haworth projection of certain compound, we have to predict the products. For all SUMO paralogs analyzed, the normally spliced transcript coding for the prototypical SUMO isoform constitutes the most abundant transcript. What are interstitial compounds. Nucleocytoplasmic fractionations aimed at determining the cellular localization of transcripts were performed using the Cytoplasmic and Nuclear RNA Purification Kit from Norgen (Norgen Biotek Corporation, Thorold, ON, Canada). Similarly, the primordial SUMO1/5 gene underwent one additional gene duplication that over time generated the current SUMO1 and SUMO5 genes. The SRA toolkit commands were incorporated into python code and the files were retrieved. Identification of the non-structural influenza A viral protein NS1A as a bona fide target of the Small Ubiquitin-like MOdifier by the use of dicistronic expression constructs. The resulting PCR products were ethanol precipitated and sequenced using the Sanger method at the Genomic Analysis Core Facility, Border Biomedical Research Center, at The University of Texas at El Paso. Knipscheer, P., van Dijk, W. J., Olsen, J. V., Mann, M. & Sixma, T. K. Noncovalent interaction between Ubc9 and SUMO promotes SUMO chain formation.

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Pozzi, B., Mammi, P., Bragado, L., Giono, L. E. & Srebrow, A. To this end, we used backbone-specific primers to amplify the backbone of the plasmid without amplifying SUMO1, and a PCR-amplified SUMO2 made using total RNA from HEK293A cells as template. We've got your back. Intriguingly, our data suggest that SUMO2 transcripts are even more abundant in tumor-derived cell lines than in normal adult tissues. Variant 1 (V1) corresponds to the normally spliced transcript, whereas the other variants correspond to alternatively spliced products.

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Finally, SUMO5 is more closely related to SUMO1 than to SUMO2/3, displaying 88% identity with SUMO1. 3) for 10 min at room temperature and proteins transferred to a PVDF membrane using the wet-transfer method at 1. Therefore, it is very likely that all SUMO alphas may still be able to interact with proteins containing classical SIMs. The absence of such amino acid residues is likely to prevent SUMO1α and SUMO2α from forming functional interactions with SAE2, thus precluding their normal activation. Classification of Elements and Periodicity in Properties. Benson, M., Iniguez-Lluhi, J.
SUMO2: Rabbit polyclonal anti-SUMO2 (Sentrin 2) from Zymed (51-9100)(Zymed Technologies, ThermoFisher Scientific, Inc. ), 1:3, 000 dilution. Ethics declarations. The tertiary structures generated for each SUMO alpha protein using the methods above were saved as "" files (protein data bank file) and viewed using UCSF Chimera, downloaded from its University of California at San Francisco repository, at Statistical analyses. HO, H, O, A CHy HC CH H. CHCH CH; 2 H, 0 excess…. The SUMO genes likely arose via successive gene duplication events, as deduced from their phylogenetic analysis and exon/intron structure 7, 8. Isabel Gutiérrez-Zubiate received support from the MERITUS program. 3) A given primer pair should amplify only one mature mRNA variant. Interestingly, our analyses showed that the nuclear retention of one specific transcript, SUMO3V2, is consistently increased upon cold-shock in both cell lines analyzed. CH;OH Br a. CH3 nCH3 NaOH Br b. КОН, …. SUMO ligases facilitate the formation of the isopeptide bond and provide some specificity to the process, as SUMO ligases are active over a relatively narrow range of protein targets. We are immensely grateful to the Campus Office of Undergraduate Research Initiatives, at The University of Texas at El Paso (UTEP) for providing access to the multitude of programs that promote and support undergraduate research activities at UTEP. The mechanism of the reaction is as follows: A: The product of the above reaction is given below, Q: Give the products of each of the following reactions: of HCI çNCH, CH, + H, 0 CH, CH, HCI + CH, OH 1.

Action of Grignard reagent. 4. none of the above. To assess the contribution of each variant to the total pool of transcripts derived from each SUMO gene, we used an RT-qPCR approach. Humans exhibit the largest prevalence of alternative splicing, with 95% of all human genes undergoing alternatively splicing 53. Pichler, A., Fatouros, C., Lee, H. & Eisenhardt, N. SUMO conjugation—a mechanistic view. Total RNA was purified using the Qiagen RNeasy Mini Kit® via the Qiashredder® method (both from QIAGEN, Inc., Redwood City, CA), as recommended by the manufacturer. Confocal microscopy.

While substantial progress has been achieved in characterizing the functions and effects associated with SUMOylation, our knowledge of the mechanisms regulating the activity of the SUMOylation system remains limited. Lee, Y. Elevated global SUMOylation in Ubc9 transgenic mice protects their brains against focal cerebral ischemic damage. Negative control samples were produced using all the ingredients minus the M-MuLV Reverse Transcriptase; nuclease-free milli-Q water was used in place of the enzyme to keep final volumes equal. Primer design approach. Oklahoma State University. While the redistribution of SUMO from one pool of targets to another is unquestionably involved in the SUMO-mediated responses to stress, findings by us and other groups support the need for additional SUMO synthesis as a likely part of the process. Such redistribution could be mediated by the activation and/or inactivation of specific sets of SUMO deconjugating enzymes and SUMO ligases.

Transfection mixes were prepared by diluting 5 μg of plasmid DNA (at a concentration of 1 μg/μL) in 380 μL of Opti-MEM™ I (Gibco™, ThermoFisher Scientific, Inc. ), and adding 15 μL of Trans-IT® LT1 transfection reagent (Mirus Bio). Hendriks, I. Site-specific characterization of endogenous SUMOylation across species and organs. An aliquot of the resulting transcript was analyzed by gel electrophoresis to ensure that the expected product size was obtained.

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