Ube2c

Manufactured by Cell Signaling Technology

Sourced in United States

UBE2C is a recombinant protein that functions as an E2 ubiquitin-conjugating enzyme. It is involved in the ubiquitination of target proteins, marking them for proteasomal degradation.

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5 protocols using ube2c

Protein Expression Analysis of MCF10A

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MCF10A protein was extracted using M-PER containing an EDTA-free Halt Protease Inhibitor Cocktail (Thermo Fisher Scientific, Massachusetts, USA), while proteins from all other cell lines were extracted using RIPA lysis buffer. Protein transfer was performed on a nitrocellulose membrane (Bio-Rad Laboratories, California, USA). Primary antibodies include: UBE2S (11878; Cell Signaling Technology, Massachusetts, USA; 1:1,000), UBE2C (14234; Cell Signaling Technology; 1:1,000), Numb (sc-136554; Santa Cruz Biotechnology, Texas, USA; 1:200), and ACTIN (3700; Cell Signaling Technology; 1:1,000). Protein was visualized with the ECL Western Blotting Detection System (PerkinElmer, USA), and a densitometry value was determined in terms of pixel intensity by ImageJ.

Guo Y., Chen X., Zhang X, & Hu X. (2023). UBE2S and UBE2C confer a poor prognosis to breast cancer via downregulation of Numb. Frontiers in Oncology, 13, 992233.

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Prostate Cancer Signaling Pathway Analysis

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Prostate cancer cells were lysed by RIPA buffer containing proteasome inhibitor cocktail (Sigma) or performed nucleocytoplasmic fractionation according to the manufacturer’s instructions (G-Biosciences). The samples were analyzed by immunoblotting with primary antibodies to: PARP (Cell Signaling Technology Cat# 9532, 1:1000), cleaved-caspase 3 (Cell Signaling Technology, Cat# 9664, 1:1000), γH2A.X (Cell Signaling Technology Cat# 2577, 1:1000), AR (Santa Cruz Biotechnology Cat# sc-7305, 1:1000), PSA (Cell Signaling Technology Cat# 5365, 1:1000), UBE2C (Cell Signaling Technology Cat# 14234, 1:200), E2F1 (Cell Signaling Technology Cat# 3742, 1:1000), FOXA1 (Cell Signaling Technology Cat# 53528, 1:1000), Ku70 (Cell Signaling Technology Cat# 4588, 1:1000), Ku80 (Cell Signaling Technology Cat# 2180, 1:1000), BRCA1 (Cell Signaling Technology Cat# 9009, 1:1000), Rad51 (Cell Signaling Technology Cat# 8875, 1:1000), Lamin B (Cell Signaling Technology Cat# 13435, 1:1000), and GAPDH (Santa Cruz Biotechnology Cat# sc-47724, 1:1000).

Lv S., Wu Z., Luo M., Zhang Y., Zhang J., Pascal L.E., Wang Z, & Wei Q. (2022). Integrated analysis reveals FOXA1 and Ku70/Ku80 as targets of ivermectin in prostate cancer. Cell Death & Disease, 13(9), 754.

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Western Blot Analysis of Signaling Pathways

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Vehicle‐ or compound‐treated cells were lysed 48 h after drug incubation with Cell Lytic buffer (Sigma; 150 mm NaCl, 0.41% bicine, 2% EDTA) supplemented with complete protease inhibitor cocktail (Roche, Basel, Switzerland) and phosphatase inhibitor cocktail (Roche). Protein concentration was quantified with BCA assay, and normalized samples were resolved with Bio‐Rad SDS/PAGE system on 8–12% protein gels. Signal detection was performed with chemiluminescence detection system (GE Healthcare, Chicago, IL, USA).
Poly(ADP‐ribose) polymerase (PARP), caspase 3, p‐ERK1/2 (Thr202/Tyr204), total ERK1/2, p‐p38 (Thr180/Tyr182), total p38, p‐BRAF (Ser445), p‐MEK1/2 (Ser217/221), total MEK1/2, SOS1, acetyl‐H3 (Lys9/14), p‐STAT3 (Ser727), total STAT3, UBE2C, HDAC1, HDAC2, HDAC3, HDAC4, HDAC5, Sirt1, β‐actin, and HRP‐conjugated anti‐rabbit were obtained from Cell Signaling (Danvers, MA, USA); SOS2 was purchased from Abcam (Cambridge, UK); FBXO3 was purchased from Sigma Aldrich (St. Louis, MO, USA); FBXW10 was purchased from Novus Biologicals (Littleton, CO, USA).

Kong L.R., Tan T.Z., Ong W.R., Bi C., Huynh H., Lee S.C., Chng W.J., Eichhorn P.J, & Goh B.C. (2017). Belinostat exerts antitumor cytotoxicity through the ubiquitin‐proteasome pathway in lung squamous cell carcinoma. Molecular Oncology, 11(8), 965-980.

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Assessing Protein Ubiquitination Dynamics

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MLN4924 and MLN7243 were purchased from MedChemExpress (Monmouth Junction, NJ, USA). Drugs were dissolved in dimethyl sulfoxide (DMSO), aliquoted, and stored at -20°C.
Primary antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA) against the following: Ubc12 (#5641S); UBE2C (#14234S); Ubc9 (#4786S); Cul3 (#2759S); Cul4A (#2699S); NAE1/APPBP1 (#14321S); Cdt1 (#8064S); p27 (#3688S); p-Chk1-Ser317 (#2344S); Chk1 (#2360S); p-Chk2-T68 (#2661S); γH2AX (#2577L); Caspase-8 (#4790S); cleaved Caspase-8 (#9748L); caspase-3 (#9662S); cleaved caspase-3 (#9661S); caspase-7 (#9492T); cleaved PARP (#5625S); BAK (#12105S); Noxa (#14766S); Puma (#4976S); BAD (#9292S); BID (#2002S); BCL-X_L (#2764S). Antibodies were purchased from Santa Cruz Biotechnology (Dallas, TX, USA) against the following: UBA3 (sc-377272); Cul1 (sc-17775); Wee1 (sc-5285); p21 (sc-271610); Chk2 (sc-9604); PARP1 (sc-7150); BAX (sc-493); BCL-2 (sc-492); MCL-1 (sc-819). Antibody against Cul4B (C9995) was from MilliporeSigma (Burlington, Massachusetts, US). Antibody against GAPDH was purchased from Beyotime Biotechnology (Shanghai, China). The secondary antibodies were purchased from Jackson ImmunoResearch Laboratories (West Grove, PA, USA).

Zhou L.N., Xiong C., Cheng Y.J., Song S.S., Bao X.B., Huan X.J., Wang T.Y., Zhang A., Miao Z.H, & He J.X. (2022). SOMCL-19-133, a novel, selective, and orally available inhibitor of NEDD8-activating enzyme (NAE) for cancer therapy. Neoplasia (New York, N.Y.), 32, 100823.

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Quantifying UBE2C Protein Expression

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Equal amounts of protein (10 µg) were loaded into Mini-Protein TGX stain-free gels (Bio-Rad) and separated using gel electrophoresis. Proteins were transferred to polyvinyl-fluoride membrane and 5% skimmed milk in Tris-buffered saline-Tween²⁰ was utilized to block unspecific binding. Primary antibody incubation was performed at room temperature for overnight (UBE2C at dilution 1:500; #14234S; Cell Signaling Technology Inc., Danvers, MA, United States). Secondary antibody incubation was carried out at room temperature for 1 h (goat anti-rabbit IgG at dilution 1:10,000; #111-035-144, Jackson ImmunoResearch, West Grove, PA, United States). Protein bands were visualized using Enhanced Chemiluminescence detection kit (Amersham ECL reagent; GE Healthcare, Barrington, IL, United States). Protein quantification was performed with Image Lab software (version 6.0, Bio-Rad) by normalizing UBE2C band intensities to amount of total protein in corresponding lane utilizing stain-free technology (Gürtler et al., 2013 (link)).

Nousiainen R., Eloranta K., Isoaho N., Cairo S., Wilson D.B., Heikinheimo M, & Pihlajoki M. (2023). UBE2C expression is elevated in hepatoblastoma and correlates with inferior patient survival. Frontiers in Genetics, 14, 1170940.

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