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Ube2c

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UBE2C is a protein that functions as an E2 ubiquitin-conjugating enzyme. It plays a role in the ubiquitin-proteasome pathway, which is responsible for the degradation of target proteins.

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Lab products found in correlation

β actin, by Abcam (3 mentions) Securin, by Abcam (2 mentions) Ube2s, by R&D Systems (2 mentions) Total s6, by Cell Signaling Technology (2 mentions) P akt ser473, by Cell Signaling Technology (2 mentions) Total akt, by Cell Signaling Technology (2 mentions) P 4ebp1, by Cell Signaling Technology (2 mentions) Puromycin dihydrochloride, by Merck Group (1 mentions) Sodium orthovanadate, by MP Biomedicals (1 mentions) Sodium pyrophosphate decahydrate, by Thermo Fisher Scientific (1 mentions) Gfp trap magnetic agarose, by Proteintech (1 mentions) Pierce high capacity streptavidin agarose, by Thermo Fisher Scientific (1 mentions) Thymidine, by Chem-Impex (1 mentions) Nocodazole, by Adipogen (1 mentions) Hygromycin b, by Thermo Fisher Scientific (1 mentions) S trityl l cysteine, by Thermo Fisher Scientific (1 mentions) Protein g sepharose, by Abcam (1 mentions) Polybrene infection transfection reagent, by Merck Group (1 mentions) Streptavidin alexafluor 568, by Thermo Fisher Scientific (1 mentions) Sodium fluoride, by Chem-Impex (1 mentions)

7 protocols using ube2c

1

In Vitro Ubiquitination Assay

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In vitro ubiquitination assays were performed as previously described with slight modifications (Oh et al., 2020) (link). The assays were carried out in a 20 μL reaction volume, and components of complete reactions included: 0.5 μL of 5 μM UBE1 (125 nM final, R&D Systems), 1 μL of 10 μM UBE2S (0.5 μM final, R&D Systems), 1 μL of 10 μM UBE2C (0.5 μM final, R&D Systems), 1 μL of 10 mg/mL His-TEV-ubiquitin (0.5 μg/μL final, prepared as described below), 2 µL of 0.5 mg/mL securin (0.5 μg/μL final, Abcam), 1 µL of 100 μM DTT, 1.5 μL of energy mix (150 mM creatine phosphate, 20 mM ATP, 20 mM MgCl2, 2 mM EGTA, pH to 7.5), 2 µL of 10X assay buffer (500 mM NaCl, 100 mM MgCl2 and 250 mM Tris pH 7.5), 5 μL of PAC/C coupled resin and 5 µL of 1X PBS. For control reactions that lacked a certain component, the mixture was supplemented accordingly with 1X PBS. Reactions were performed at 30 ºC with shaking for 30 min and stopped by addition of 6X Laemmli Buffer and incubation at 95 ºC for 5 min. Samples were analyzed by SDS-PAGE and Western blot.
Cao X., Shah A.S., Sanford E.J., Smolka M.B., & Baskin J.M. (2022). Proximity labeling reveals spatial regulation of the anaphase-promoting complex/cyclosome by a microtubule adaptor.
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2

Comprehensive Cell Signaling Assays

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Lipofectamine 2000, Invitrogen (11668019); Lipofectamine RNAiMAX, Invitrogen (13778150); Polybrene Infection/Transfection Reagent, Sigma-Aldrich (TR-1003); GFP-Trap magnetic agarose, ChromoTek (gtma-20); Trypsin Gold Mass Spectrometry Grade, Promega (V5280); Protein G Sepharose, BioVision (6511); Thymidine, Chem-Impex (00306); Propidium iodide, Sigma-Aldrich (P4170); RNase A, Research Products International (R21750); Nocodazole, AdipoGen (AG-CR1-0019); (+)-S-Trityl-L-cysteine, Alfa Aesar (L14384); Puromycin dihydrochloride, Sigma-Aldrich (P8833); Blasticidin S hydrochloride, 10 mg/ml in HEPES buffer, Alfa Aesar (J67216); Hygromycin B, 50 mg/mL in PBS, Invitrogen (10687010); cOmplete Protease Inhibitor Cocktail, Roche (5056489001); Sodium β-glycerophosphate pentahydrate, Alfa Aesar (L03425); Sodium fluoride, Chem-Impex (01523); Sodium pyrophosphate decahydrate, Fisher (S390); Sodium orthovanadate, MP Bio (0215966410); UBE1, R&D Systems (E-304); UBE2C, R&D Systems (E2-654); UBE2S, R&D Systems (E2-690); securin, Abcam (ab87664); D-biotin, Chem-Impex (00033); Streptavidin Alexa Fluor 568, Invitrogen (S11226); Pierce™ High Capacity Streptavidin Agarose, Thermo Fisher (20359); Streptavidin-conjugated HRP, GeneTex (GTX85912); ProLong™ Diamond Antifade Mountant with DAPI, Thermo Fisher (P36971); Clarity™ Western ECL Substrate, Bio-Rad (1705061).
Cao X., Shah A.S., Sanford E.J., Smolka M.B., & Baskin J.M. (2022). Proximity labeling reveals spatial regulation of the anaphase-promoting complex/cyclosome by a microtubule adaptor.
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3

Prostate Cancer Cell Signaling Assay

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LNCaP95 cells were serum-starved for 24hr, followed by treatment with DMSO, EPI-002 (25uM), enzalutamide (10 uM), BEZ235 (15nM) or a combination for 1hr prior to addition of R1881 or EtOH for 48hr. Antibodies used were: AR (1:1000; Santa Cruz), AR-V7 (1:400; Precision), p110α (1:500; BD Bioscience), p110β (1:1000; abcam), p100γ (1:1000; abcam), p110δ (1:1000; abcam), UBE2C (Boston Biochem; 1:1000), PTEN (1:1000), pS6 (1:2000), pAktThr308 (1:1000), pAktSer473 (1:2000), p4EBP1 (1:1000), total-Akt (1:1000), total-S6 (1:1000), total-4EBP1 (1:1000), pERK/MAPK (1:1000), total-ERK/MAPK (1:1000) from Cell Signaling technology (Danvers, MA). β-actin (1:10,000, Abcam) was used as a loading control.
Kato M., Banuelos C.A., Imamura Y., Leung J.K., Caley D.P., Wang J., Mawji N.R, & Sadar M.D. (2015). Co-targeting Androgen Receptor Splice Variants and mTOR signaling pathway for the Treatment of Castration-Resistant Prostate Cancer. Clinical cancer research : an official journal of the American Association for Cancer Research, 22(11), 2744-2754.
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4

Evaluation of Androgen Receptor Protein

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Immunohistochemistry was performed as previously described (22 (link), 23 (link)). For analysis of AR protein, concentrations of lysates of homogenized LNCaP95 and PC3 xenografts were measured by bicinchoninic acid assay after albumin depletion. Proteins (20 μg) were resolved on a NuPAGE 4%–15% Bis Tris gradient gel, transferred to nitrocellulose membrane, and probed for AR species using antibodies to the AR NTD and AR-V7. LNCaP95 cells (250,000 cells/well) were seeded in a 6-well plate for 48 hours and serum-starved for 24 hours, followed by treatment with DMSO, enzalutamide (5 μM), EPI-002 (25 μM), or I-EPI-002 (5 μM) for 48 hours. Cells were harvested, and whole-cell lysate (10 to 15 μg) was subjected to SDS-PAGE. Antibodies used were as follows: AR N-20 (sc-816, Santa Cruz Biotech Inc.); AR-V7 (AG10008, Precision Antibody); UBE2C (A-650, BostonBiochem); and cyclinD1, cyclinD3, p27kip1, CDK2, CDK4, and CDK6 from the Cell Cycle Regulation Sampler Kit (9932 from Cell Signaling Technology). β-Actin (ab6276, Abcam) was used as a loading control.
Imamura Y., Tien A.H., Pan J., Leung J.K., Banuelos C.A., Jian K., Wang J., Mawji N.R., Fernandez J.G., Lin K.S., Andersen R.J, & Sadar M.D. (2016). An imaging agent to detect androgen receptor and its active splice variants in prostate cancer. JCI Insight, 1(11), e87850.
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5

Western Blot Protein Analysis Protocol

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Western blots were performed as previously described in Reference [26 (link)]. The primary antibodies used were: AR (1:1000; Santa Cruz Biotechnology), AR-V7 (1:400; Precision), GR (1:1000; BD transduction laboratories), PSA (1:1000; Santa Cruz Biotechnology), FKBP5 (1:1000; Santa Cruz Biotechnology), UBE2C (1:1000; Boston Biochem), NSE (1:1000; Merck), Mdr-1 (1:1000; Santa Cruz), Aurora A (1:1000), BRN-2 (1:1000), total-STAT3 (1:1000), p-STAT3Tyr705 (1:1000), total-AKT (1:1000), p-AktSer473 (1:1000), total-S6 (1:1000), p-S6 (1:2500), total-p44/42MAPKErk1/2 (1:1000), p-p44/42MAPKErk1/2 (1:1000), 110α (1:1000), 110β (1:1000), 110γ (1:1000), PI3KClass III; (1:1000), p85 (1:1000), 4EBP1 (1:1000), and p-4EBP1 (1:1000), from Cell Signaling Technology. Beta-actin (1:1000, Abcam and Cell Signaling Technology) was used as a loading control.
Shimizu Y., Tamada S., Kato M., Hirayama Y., Takeyama Y., Iguchi T., Sadar M.D, & Nakatani T. (2018). Androgen Receptor Splice Variant 7 Drives the Growth of Castration Resistant Prostate Cancer without Being Involved in the Efficacy of Taxane Chemotherapy. Journal of Clinical Medicine, 7(11), 444.
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6

Protein Analysis of Cell Lysates

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The cells were harvested and whole‐cell lysates were prepared using lysis buffer (50 mM HEPES [pH 7.5], 150 mM NaCl, 5 mM EDTA, 1% NP‐40, and 0.65% CHAPS containing 1 mM PMSF and a protease inhibitor cocktail). Sample preparation and Western blot analysis were carried out as previously described18 using antibodies against the following proteins: Hsp70, AR (N‐20), β‐actin, c‐Jun, and Twist (Santa Cruz Biotechnology, Dallas, TX, USA); Hsp90, PSA, YB‐1, phospho‐YB‐1, and phospho‐Foxo3a (Cell Signaling Technology, Danvers, MA, USA); AR‐V7 (Precision Antibody, Columbia, MD, USA); UBE2C (BostonBiochem, Cambridge, MA, USA); FKBP5, CREB, and Sp1 (Gene Tex, CA, USA); laminB1 (Abcam, Cambridge, UK); Foxo3a (Upstate, Temecula, CA, USA); α‐tubulin (Calbiochem, San Diego, CA, USA); and HRP‐conjugated secondary antibodies (GE Healthcare, Little Chalfont, UK).
Kita K., Shiota M., Tanaka M., Otsuka A., Matsumoto M., Kato M., Tamada S., Iwao H., Miura K., Nakatani T, & Tomita S. (2017). Heat shock protein 70 inhibitors suppress androgen receptor expression in LNCaP95 prostate cancer cells. Cancer Science, 108(9), 1820-1827.
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7

Amino Acid Transporter Regulation Assay

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Cells were seeded at a density of 2 ×
105 in 6-well plates, allowed to adhere overnight, before
incubation with DMSO, 10 mM BCH, 50 μM ESK242, or 50 μM
ESK246 for 6 h or 3 d. Cells were lysed by the addition of lysis buffer
(200 μL) with protease inhibitor Cocktail III (Bioprocessing
Biochemical) and 1 mM Na3VO4 (Sigma). Equal
protein (micro-BCA method; Pierce, IL) was loaded on 4–12%
gradient gels (Life Technologies), electrophoresed, and transferred
to PVDF membrane. The membrane was blocked with 2.5% (w/v) BSA in
PBS-Tween20, and incubated with the primary and secondary antibodies.
The secondary HRP-labeled antibodies were detected using enhanced
chemiluminescence reagents (Pierce) on a Kodak Imager (Kodak). Antibodies
used in this study were against LAT1 (Cosmo Bio), LAT3 (a kind gift
from Kunimasa Yan, Kyorin University, Tokyo, Japan), α-tubulin
(Santa Cruz), p-p70S6K, p70S6K, (Cell Signaling), UBE2C (Boston Biochem),
CDK1 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH; Abcam).
Horseradish peroxidase-conjugated donkey antimouse IgG, donkey antirabbit
IgG, and goat antimouse IgM were used as secondary antibodies (Millipore).
Wang Q., Grkovic T., Font J., Bonham S., Pouwer R.H., Bailey C.G., Moran A.M., Ryan R.M., Rasko J.E., Jormakka M., Quinn R.J, & Holst J. (2014). Monoterpene Glycoside ESK246 from Pittosporum Targets LAT3 Amino Acid Transport and Prostate Cancer Cell Growth. ACS Chemical Biology, 9(6), 1369-1376.
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