Ube2c

Manufactured by Abcam

Sourced in United States

UBE2C is an enzyme that plays a role in the ubiquitination process, which targets proteins for degradation. It is involved in the regulation of the cell cycle.

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Lab products found in correlation

Pvdf membrane, by Merck Group (2 mentions) Involucrin, by Santa Cruz Biotechnology (1 mentions) Np40 lysis buffer, by Thermo Fisher Scientific (1 mentions) Hrp conjugated secondary antibody, by Merck Group (1 mentions) Akt inhibitor ly294002, by Cell Signaling Technology (1 mentions) β catenin, by Santa Cruz Biotechnology (1 mentions) Ecl substrate, by Thermo Fisher Scientific (1 mentions) E cadherin, by Santa Cruz Biotechnology (1 mentions) Enhanced chemiluminescence detection reagent, by Cytiva (1 mentions) Anti abcg2, by Santa Cruz Biotechnology (1 mentions) Ercc1, by Santa Cruz Biotechnology (1 mentions) Cleaved capase 3, by Abcam (1 mentions) Vimentin, by Abcam (1 mentions) E cadherin, by Abcam (1 mentions) Beta catenin, by Roche (1 mentions) Vectra polaris, by PerkinElmer (1 mentions) Ido 1, by Merck Group (1 mentions) Granzyme b, by Cell Signaling Technology (1 mentions) Idh1 r132h, by Dianova (1 mentions) P gsk 3β, by Abcam (1 mentions)

Close spelling variants of this product

Anti-UBE2C (2 citations)

5 protocols using ube2c

Immunoblotting Analysis of Stemness and EMT Markers

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Cell lysates were prepared after 48 hrs of transfection in NP-40 lysis buffer (Invitrogen) and protease inhibitor cocktail (1X). Equivalent amounts of denatured protein samples were subjected to SDS-PAGE (8%-10%), separated by size and transferred on to PVDF membrane (Millipore, Billerica, USA). Antibodies used for immuno-blotting were polyclonal β-catenin, E-cadherin, CD44 and CD24, Involucrin (Santa Cruz Biotechnology, CA, USA), polyclonal Oct4 and Sox2 (Abcam), polyclonal C-myc, Akt and phospho-Akt (Cell Signaling Technology, USA) and UBE2C (Abcam). Bands were obtained using ECL substrate (Thermo Scientific, USA) from HRP-conjugated secondary antibody (Sigma). Proteasome Inhibitor MG132 (Calbiochem) and Akt inhibitor LY294002 (Cell signaling Technology, USA) were both used at a concentration of 50 µM. Transfected cells were treated for 4 hours before harvesting. Band intensities of each protein were analyzed by ImageJ to obtain densitometric values for their quantification. These were normalized to β-actin for individual experimental sets and fold change calculated. All the histograms were expressed as means ± S.D. of three different experiments and p values computed in GraphPad Prism 5 (Student’s two tailed t test).

Ghuwalewala S., Ghatak D., Das S., Roy S., Das P., Butti R., Gorain M., Nath S., Kundu G.C, & Roychoudhury S. (2021). MiRNA-146a/AKT/β-Catenin Activation Regulates Cancer Stem Cell Phenotype in Oral Squamous Cell Carcinoma by Targeting CD24. Frontiers in Oncology, 11, 651692.

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Western Blot Analysis of Protein Expression

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For western blotting analysis, proteins of each group cells were denatured at 100°C boiled 5 min with SDS loading buffer. The proteins were transferred to PVDF transfer membrane. Membranes were incubated with the indicated antibodies overnight at 4°C followed by immunoblotting analysis. Proteins were detected using enhanced chemiluminescence detection reagents (Amersham). Tubulin was internal control. The primary antibodies used in this study were 1:1000 rabbit anti-ABCG2 and ERCC1 (Santa Cruz, Dallas, TX, USA), 1:1000 antibody of Tubulin, UBE2C, ZEB1, ZEB2, vimentin, E-cadherin, and cleaved capase-3 (Abcam, Cambridge, UK). The gray intensity analysis of western blotting images was carried out by ImageJ software.

Wu Y., Jin D., Wang X., Du J., Di W., An J., Shao C, & Guo J. (2019). UBE2C Induces Cisplatin Resistance via ZEB1/2-Dependent Upregulation of ABCG2 and ERCC1 in NSCLC Cells. Journal of Oncology, 2019, 8607859.

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Immunohistochemical Profiling of Tumor Markers in Merkel Cell Carcinoma

Cited 1 time

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Immunohistochemistry for granzyme B (Cell Signaling Technology), IDO-1 (Sigma Aldrich), UBE2C (Abcam), beta catenin (Ventana), and IDH1 R132H (Dianova) expression was performed using the conditions outlined in Table S5. granzyme B was selected for validation due to common use of this marker in diagnostic laboratories, after confirmation that GZMA and GZMB transcript expression were closely correlated in previously published MCC expression profiles [17 (link)]. Potential prognostic markers (granzyme, IDO1, UBE2C) were evaluated using previously described tissue microarrays and whole sections on primary tumors,[26 (link)] alongside additional primary tumor cases, to achieve adequate statistical power. Immunostained slides were scanned at 20x magnification on a Vectra Polaris (PerkinElmer). Scoring was performed on representative digitized tumor fields selected by a board-certified dermatopathologist (P.W.H.) using the Positive Cell Detection analysis in QuPATH software to quantitate either density of positive inflammatory cells (granzyme, IDO1) or H-score expression on tumor cells (UBE2C, beta-catenin). Beta-catenin and IDH1 R132H immunohistochemistry were performed on representative mutant and wild type tumors.

Harms K.L., Zhao L., Johnson B., Wang X., Carskadon S., Palanisamy N., Rhodes D.R., Mannan R., Vo J.N., Choi J.E., Chan M.P., Fullen D.R., Patel R.M., Siddiqui J., Ma V.T., Hrycaj S., McLean S.A., Hughes T.M., Bichakjian C.K., Tomlins S.A, & Harms P.W. (2021). Virus-positive Merkel cell carcinoma is an independent prognostic group with distinct predictive biomarkers. Clinical cancer research : an official journal of the American Association for Cancer Research, 27(9), 2494-2504.

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Western Blot Analysis of EMT Markers

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UBE2C, AURKA, β-catenin, AKT1, p-AKT1, GSK-3β, p-GSK-3β, Slug, Snail and Twist antibodies were purchased from Abcam (Cambridge, UK). E-cadherin, N-cadherin and p-AURKA antibodies were purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA). The GAPDH antibody was purchased from Zhongshan Golden Bridge Biotechnology (Beijing, China). Western blot analysis reagents were purchased from Sigma, PVDF membranes were purchased from Millipore Corp. (Bedford, MA, USA) and RIPA lysis buffer was purchased from Beijing Taike Biotechnology. Lentiviruses containing an UBE2C and AURKA inhibitor sequences (Lenti-si-UBE2C and Lenti-si-AURKA) or negative control (Lenti-NC) were obtained from Shanghai GeneChem Co., Ltd. (Shanghai, China).

Wang R., Song Y., Liu X., Wang Q., Wang Y., Li L., Kang C, & Zhang Q. (2017). UBE2C induces EMT through Wnt/β-catenin and PI3K/Akt signaling pathways by regulating phosphorylation levels of Aurora-A. International Journal of Oncology, 50(4), 1116-1126.

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In vitro Ubiquitination Assay for RNF8

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For in vitro ubiquitination assay, 1 μg histone H2A (New England Biolabs) or 1 μg H2A/H2B dimer or 2 μg mononucleosome was incubated with 50 ng E1 (Boston Biochem), 50 or 100 ng of each of Ube2S and Ube2C (Abcam), 2 μg ubiquitin (Boston Biochem) and purified HA-Flag-RNF8 in 20 μl reaction mixture buffer (50 mM Tris, 5 mM KCl, 5 mM MgCl2, 0.5 mM DTT, 200 mM Creatine phosphate (Calbiochem), 2 μg/μl Creatine Phosphokinase (Calbiochem), 2 mM ATP(New England Biolabs) at 32°C for 2 hr. The reaction was stopped by addition of sample buffer and resolved by SDS-PAGE. HA-Flag-RNF8 was purified from HEK293T cells expressing HA-Flag-tagged RNF8. Flag immunoprecipitation was carried out by incubating Flag-beads with cell lysates overnight at 4°C with gentle agitation. The beads were then washed with NETN buffer followed by elution with 3X Flag peptide (Sigma). For RNF8 autoubiquitination assay, HA-Flag-RNF8 was incubated with purified ubiquitin (2 μg), E1 activating enzyme (50 ng), 50 or 100 ng of each of Ube2S and Ube2C in the reaction mixture buffer at 32°C for 2 hr. For recombinant GST-RNF8 in vitro autounbiquitination assay, 500–1000 ng of purified GST-RNF8 was used Reaction was stopped by addition of 2X sample loading buffer and analyzed by western blot with FK2 antibody.

Paul A, & Wang B. (2017). RNF8- and Ube2S-dependent ubiquitin lysine 11-linkage modification in response to DNA damage. Molecular cell, 66(4), 458-472.e5.

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