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E2 conjugating enzyme ubch5c

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E2-conjugating enzyme UbcH5c is a recombinant protein that functions as an E2 ubiquitin-conjugating enzyme. It is involved in the ubiquitination pathway, playing a role in the covalent attachment of ubiquitin to target proteins.

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Lab products found in correlation

Adenosine triphosphate (atp), by R&D Systems (3 mentions) Ha ubiquitin, by R&D Systems (3 mentions) E1 ubiquitin activating enzyme ube1, by R&D Systems (3 mentions) Usinglipofectamine plus, by Thermo Fisher Scientific (1 mentions) Ha ub, by R&D Systems (1 mentions) Flag peptide, by Merck Group (1 mentions) Anti ha, by Roche (1 mentions)

4 protocols using e2 conjugating enzyme ubch5c

1

Assaying Ptch1 Ubiquitination In Vivo and In Vitro

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To assay for Ptch1 ubiquitination in vivo,
Smurf1−/−/Smurf2flox/floxMEFs were infected with either Ad-GFP or Ad-Cre adenovirus for 24 hr, then
transfected with Ptch1-FLAG or Ptch1Δ2PY-FLAG along with HA-Ub using
Lipofectamine Plus (Invitrogen). The cells were lysed 24 hr later and Ptch1 and
its mutant were isolated with anti-FLAG agarose beads and resolved by SDS-PAGE
on 6% or 4–12% gradient gels. The ubiquitinated Ptch1 was then detected
with anti-HA (Roche-Shanghai, China). To assay for Ptch ubiquitination in vitro,
an ubiquitination assay was modified from a previously described procedure
(Tang et al., 2011 (link)). Ptch1-FLAG was
captured from transfected HEK293 cell lysates using anti-FLAG agarose. After a
thorough wash, the Ptch1-bound agarose was divided into three aliquots. Empty
anti-FLAG agarose was used as a control. The in vitro ubiquitination assay was
performed by incubating either Ptch1-bound agarose or control agarose at
37°C for 1 hr with ubiquitin-activating enzyme UBE1, E2-conjugating enzyme
UbcH5c, HA-Ub and ATP (all from Boston Biochem, Cambridge, MA) in the presence
or absence of purified His6-Smurf2 or His6-Smurf2CG. After the incubation, the
supernatant was removed, the agarose thoroughly washed, and the Ptch1-FLAG
eluted using the FLAG peptide (Sigma). The eluted fraction was then subjected to
Western blot analysis.
Yue S., Tang L.Y., Tang Y., Tang Y., Shen Q.H., Ding J., Chen Y., Zhang Z., Yu T.T., Zhang Y.E, & Cheng S.Y. (2014). Requirement of Smurf-mediated endocytosis of Patched1 in sonic hedgehog signal reception. eLife, 3, e02555.
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2

In vitro Ubiquitination Assay of TRIM33

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FLAG-TRIM33 or its CA mutant was captured from transfected HEK 293T cell lysates by anti-FLAG agarose. After a thorough wash, the agarose was divided into aliquots. Empty anti-FLAG agarose was used as control. The in vitro ubiquitination assay was performed by incubating TRIM33-bound, mutant TRIM33-bound, or control agarose at 37°C for 1 h with E1 ubiquitin-activating enzyme UBE1, E2-conjugating enzyme UbcH5c, HA-ubiquitin, and ATP (all from Boston Biochem) in the presence or absence of purified GST–β-catenin WT or mutant or active PKCδ. The supernatant was removed after the assay and the agarose was thoroughly washed and analysed by IB.
Xue J., Chen Y., Wu Y., Wang Z., Zhou A., Zhang S., Lin K., Aldape K., Majumder S., Lu Z, & Huang S. (2015). Tumour suppressor TRIM33 targets nuclear β-catenin degradation. Nature communications, 6, 6156.
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3

Ubiquitination Assay of KDM4C

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Flag-KDM4C was captured from Flag-KDM4C plasmid-transfected HEK293T cell lysates by anti-Flag agarose beads. Empty anti-Flag agarose beads were used as a control. The in vitro ubiquitination assay was performed by incubating KDM4C or control agarose beads at 37°C for 1 hour with E1 ubiquitin-activating enzyme UBE1, E2-conjugating enzyme UbcH5c, HA-ubiquitin, and adenosine triphosphate (all from Boston Biochem) in the presence or absence of recombinant β-TrCP protein (Creative Biomart, BTRC-2545M). Then the supernatant was removed, and the beads were thoroughly washed and boiled in 1× loading buffer, followed by Western blot analysis with the indicated antibodies.
Chen Y., Fang R., Yue C., Chang G., Li P., Guo Q., Wang J., Zhou A., Zhang S., Fuller G.N., Shi X, & Huang S. (2019). Wnt-Induced Stabilization of KDM4C Is Required for Wnt/β-Catenin Target Gene Expression and Glioblastoma Tumorigenesis. Cancer research, 80(5), 1049-1063.
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4

In vitro Ubiquitination Assay of TRIM33

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FLAG-TRIM33 or its CA mutant was captured from transfected HEK 293T cell lysates by anti-FLAG agarose. After a thorough wash, the agarose was divided into aliquots. Empty anti-FLAG agarose was used as control. The in vitro ubiquitination assay was performed by incubating TRIM33-bound, mutant TRIM33-bound, or control agarose at 37°C for 1 h with E1 ubiquitin-activating enzyme UBE1, E2-conjugating enzyme UbcH5c, HA-ubiquitin, and ATP (all from Boston Biochem) in the presence or absence of purified GST–β-catenin WT or mutant or active PKCδ. The supernatant was removed after the assay and the agarose was thoroughly washed and analysed by IB.
Xue J., Chen Y., Wu Y., Wang Z., Zhou A., Zhang S., Lin K., Aldape K., Majumder S., Lu Z, & Huang S. (2015). Tumour suppressor TRIM33 targets nuclear β-catenin degradation. Nature communications, 6, 6156.
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