Cul4a

Manufactured by Cell Signaling Technology

Sourced in United States

CUL4A is a protein that is a component of the Cullin-RING E3 ubiquitin ligase complex. This complex is involved in the ubiquitination and subsequent degradation of target proteins.

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Lab products found in correlation

Cul4b, by Proteintech (2 mentions) Ube2m, by Santa Cruz Biotechnology (2 mentions) Cul 5, by Santa Cruz Biotechnology (2 mentions) Cleaved caspase 3, by Cell Signaling Technology (2 mentions) β actin, by Merck Group (2 mentions) Cyclin k, by Fortis Life Sciences (1 mentions) Cdk12, by Cell Signaling Technology (1 mentions) Ubiquityl histone h2a k119, by Cell Signaling Technology (1 mentions) Vinculin, by Santa Cruz Biotechnology (1 mentions) Cdk13, by Fortis Life Sciences (1 mentions) Alexa fluor 488, by Thermo Fisher Scientific (1 mentions) Anti klf2, by Merck Group (1 mentions) 0.22 mm nc membrane, by Merck Group (1 mentions) Anti gapdh, by Cell Signaling Technology (1 mentions) Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions) Odyssey clx infrared imager, by LI COR (1 mentions) Anti rabbit irdye 800, by LI COR (1 mentions) Anti mouse irdye 680, by LI COR (1 mentions) Aldoa, by Abcam (1 mentions) Cell culture medium, by Thermo Fisher Scientific (1 mentions)

10 protocols using cul4a

Western Blot Analysis of Cullin Proteins

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PBS-washed cell pellets were lysed in 50mM Tris pH 7.9, 8M Urea and 1% CHAPS and incubated with shaking at 4ºC for at least 30min. 20μg of supernatants were run and transferred for detection. Antibodies used: CUL1 (1:1000, Santa Cruz Biotechnology, sc-1276), CUL2 (1:500, Sigma-Aldrich, SAB2501565-100), CUL3 (1:1000, Cell Signaling Technology, 2759), CUL4A (1:1000, Cell Signaling Technology, 2699S), CUL4B (1:1000, Proteintech, 12916-1-AP), CUL5 (1:1000, Santa Cruz Biotechnology, sc-373822), UBE2M (1:1000, Santa Cruz Biotechnology, sc-390064), DDB1 (1:1000, Cell Signaling Technology, 5428S), cyclin K (1:5000, Bethyl, A301-939A), CDK12 (1:1000, Cell Signaling Technology, 11973S), CDK13 (1:1000, Bethyl, A301-458A), RBM39 (1:500, Santa Cruz Biotechnology sc-376531), V5 (1:1000 Cell Signaling Technology, 13202), Ubiquityl-Histone H2A (K119) (1:1000, Cell Signaling, 8240-20), MLH1 (1:1000, Cell Signaling Technology, 3515T). ACTIN (1:10000, Sigma-Aldrich, A5441), VINCULIN (1:1000, Santa Cruz Biotechnology, sc-25336). Secondary antibodies anti-mouse/rabbit/goat (1:10000, Jackson ImmunoResearch 115-035-003, 111-035-003 and 705-035-003) and anti-rabbit Alexa Fluor 488 (1:1000, Invitrogen, A21441).

Mayor-Ruiz C., Bauer S., Brand M., Kozicka Z., Siklos M., Imrichova H., Kaltheuner I.H., Hahn E., Seiler K., Koren A., Petzold G., Fellner M., Bock C., Müller A.C., Zuber J., Geyer M., Thomä N.H., Kubicek S, & Winter G.E. (2020). Rational discovery of molecular glue degraders via scalable chemical profiling. Nature chemical biology, 16(11), 1199-1207.

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Western Blot Protein Analysis

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Cells were harvested, and protein was extracted from transfected cells and quantified as previously described⁴¹ (link) using 12% or 4%–20% polyacrylamide gradient SDS gel. Cell protein lysates were separated by 10% SDS-PAGE, transferred to 0.22 mm NC membranes (Sigma), and incubated with specific antibodies. GAPDH antibody was used as control, and anti-GAPDH and CUL4A (1:1,000) were purchased from Cell Signaling Technology; anti-KLF2 was purchased from Sigma. Protein detection was performed with Super Signal Chemiluminescence Substrate (Pierce).

Lian Y., Yan C., Xu H., Yang J., Yu Y., Zhou J., Shi Y., Ren J., Ji G, & Wang K. (2018). A Novel lncRNA, LINC00460, Affects Cell Proliferation and Apoptosis by Regulating KLF2 and CUL4A Expression in Colorectal Cancer. Molecular Therapy. Nucleic Acids, 12, 684-697.

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Estrogen Regulation of Muscle Cell Proteins

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Primary human muscle cell line that was derived from the quadriceps muscle biopsy of a 5‐day‐old female infant (Edom et al., 1994) was kindly provided by Profs Moyly and Buttler‐Browne (INSERM, Paris, France). Proliferating mononuclear myoblasts were differentiated for 5 days to form multinuclear myotubes in E₂ free environment, before exposing them to 10 nm E₂, 100 nm E₂ or mock for 6 h. All experiments were carried out in triplicate. Semi‐quantitative immunoblotting analysis was carried out to quantitate the protein expression of CUL4A, MT‐CO2, LDHA, ALDOA, and PKM. TUBA was used for normalization. The blots were scanned and quantified using Odyssey CLX Infrared Imager of Li‐COR and manufacturer's software. Cell culture mediums, fetal bovine serum, and antibiotics were obtained from Life Technologies, Inc. (Carlsbad, CA, USA) while insulin from Sigma‐Aldrich. CUL4A, MT‐CO2, and LDHA antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA), ALDOA and PKM from Abcam (Cambridge, UK) and TUBA from Sigma‐Aldrich. As a secondary antibodies, anti‐rabbit IR Dye 800 or anti‐mouse IR Dye 680 (LI‐COR Biosciences, Lincoln, NE, USA) was used.

Laakkonen E.K., Soliymani R., Karvinen S., Kaprio J., Kujala U.M., Baumann M., Sipilä S., Kovanen V, & Lalowski M. (2017). Estrogenic regulation of skeletal muscle proteome: a study of premenopausal women and postmenopausal MZ cotwins discordant for hormonal therapy. Aging Cell, 16(6), 1276-1287.

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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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Analysis of Cullin-RING Ubiquitin Ligases

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Cells after indicated treatment were collected, lysed and examined by standard western blot procedures. SAG monoclonal Ab was raised against the RING domain (AA44-113) [42] (link). The other antibodies were purchased from a variety of vendors as follows: CUL1^CTD (Proteintech, 12895-1-AP), CUL1 (Santa Cruz, SC-11384), CUL5^CTD (Sigma-Aldrich, AV35127), CUL5 (Santa Cruz, sc-373822), CUL3 (Cell signaling, 2759S), CUL2 (abcam, ab166917), CUL4A (Cell signaling, 2699S), CUL4B (Proteintech, 12916-1-AP), RBX1 (Cell signaling, 11922S), NEDD8 (abcam, ab81264), UBA3 (abcam, ab124728), NAE1 (Cell signaling, 14321S), UBE2F (Santa Cruz, sc-398668), UBE2M (Santa Cruz, sc-390064), p21 (Cell signaling, 2947S), p27 (Cell signaling, 2552S), MCL1 (Cell signaling, 5453S), NRF2 (Santa Cruz, sc-722), CDT1 (Santa Cruz, sc-28262), NOXA (EMD Millipore, OP180), and β-Actin (Sigma-Aldrich, A5441).

Yu Q., Hu Z., Shen Y., Jiang Y., Pan P., Hou T., Pan Z.Q., Huang J, & Sun Y. (2020). Gossypol inhibits cullin neddylation by targeting SAG-CUL5 and RBX1-CUL1 complexes. Neoplasia (New York, N.Y.), 22(4), 179-191.

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Comprehensive Western Blot and Immunostaining Protocol

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Total tau TAU5 for western blotting (Invitrogen, Rockford, IL), total tau TAU5 for IP (AbCam, Cambridge, MA), total tau K9JA for IF (Dako/Agilent, Santa Clara, CA), P-tau Ser396 (Invitrogen), P-tau PHF-1 (kindly provided by Dr. Peter Davies, Albert Einstein College of Medicine, NY). Neuronal marker MAP2 (Chemicon/Millipore). CRL4^CRBN E3 Ligase components DDB1 (AbCam), CUL4A (Cell Signaling Technology), and CRBN (ProteinTech, Rosemont, IL). Anti-Ubiquitin, clone Ubi-1 (Millipore, Darmstadt, Germany). Internal controls GAPDH (Abcam) and β-Actin (Sigma). Nuclear stain Hoechst 33342 (Invitrogen).

Silva M.C., Ferguson F.M., Cai Q., Donovan K.A., Nandi G., Patnaik D., Zhang T., Huang H.T., Lucente D.E., Dickerson B.C., Mitchison T.J., Fischer E.S., Gray N.S, & Haggarty S.J. (2019). Targeted degradation of aberrant tau in frontotemporal dementia patient-derived neuronal cell models. eLife, 8, e45457.

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Protein Extraction and Western Blot Analysis

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Total protein was lysed from cells and tissues utilizing RIPA buffer, and its concentrations were measured using a BCA kit (Beyotime, China). Equal amounts of protein were separated by SDS-PAGE, then electro-transferred onto nitrocellulose (NC) membrane (Pall, USA). The membrane was blocked with 5% skimmed milk for 2h at room temperature (RT). Blots were incubated with primary antibodies overnight at 4℃, washed with 1×TBST (3×10 min), and subsequently incubated with secondary antibodies for 2h at RT. Protein bands were exposed with an enhanced chemiluminescence reagent (Beyotime, China), and visualized by gel analysis imaging system (Tanon, China). The following antibodies were used: S100A16, pLATS1 and pMST1/2 (Proteintech, China), β-Actin (Bioss, China), LATS1, MST1, MST2, SAV, pMOB, MOB, pYAP, YAP/TAZ, Ub, CUL4A (Cell Signaling Technology, USA), and DCAF1, DDB1 (Abcam, UK).

Hu Y., Zhang R., Lu S., Zhang W., Wang D., Ge Y., Jiang F., Qin X, & Liu Y. (2023). S100 Calcium Binding Protein A16 Promotes Cell Proliferation by triggering LATS1 ubiquitin degradation mediated by CUL4A ligase to inhibit Hippo pathway in Glioma development. International Journal of Biological Sciences, 19(7), 2034-2052.

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Antibody Generation and Protein Purification Protocol

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BRWD2/PHIP antibodies (Shilatifard, nos. 833 and 834) were raised against a protein fragment spanning BRWD2/PHIP amino acids 1681–1821 (sequence ID NP_060404.4). Drosophila dBRWD3 antibody (Shilatifard, no. 385) was raised against amino acids 976–1495 (sequence ID NP_732982.1). Proteins were expressed from the pET16 plasmid in Rosetta2 E. coli cells and purified using Ni-NTA agarose resin (Qiagen). Rabbit immunization was performed by Pocono Rabbit Farm and Laboratory. Other antibodies used in this study were H3K4me1 (Shilatifard Laboratory, no. 24), H3K4me2 (Shilatifard Laboratory, no. 27), H3K4me3 (Shilatifard Laboratory, no. 680), H3K27ac (Cell Signaling Technology, no. 8137), H2A.Z (Cell Signaling Technology, no. 2718), H3K27me3 (Shilatifard Laboratory, no. 67), DDB1 (Cell Signaling Technology, no. 6998), CUL4A (Cell Signaling Technology, no. 2699), CHD4 (Cell Signaling Technology #12011), PHF6 (Santa Cruz Biotechnology, no. sc-365237), β-Tubulin (E7, Developmental Studies Hybridoma Bank), Histone H3 (Shilatifard Laboratory, no. 42), and Flag-M2 (Sigma, no. F1804).

Morgan M.A., Rickels R.A., Collings C.K., He X., Cao K., Herz H.M., Cozzolino K.A., Abshiru N.A., Marshall S.A., Rendleman E.J., Sze C.C., Piunti A., Kelleher N.L., Savas J.N, & Shilatifard A. (2017). A cryptic Tudor domain links BRWD2/PHIP to COMPASS-mediated histone H3K4 methylation. Genes & Development, 31(19), 2003-2014.

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Immunoblot Analysis of Signaling Pathways

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Antibodies against p-ULK1(S757), ULK1, p-4EBP1(S65)/(T37), 4EBP1, p-S6K(T389), S6K, p-eIF4E(S209), eIF4E, p-S6(SS240/4), S6, DEPTOR, p-AKT(S473)/(T308), AKT, MNK1, p-MNK1(TT197/202), mTOR, Raptor, p-PRAS40(T246), PRAS40, p-ERK1/2(T202/Y204), ERK1/2, GβL, eIF4G1, DDB1, ATM, HSP90, βTRC, Cul4A, RBX1, DDB2, Rictor, Sin1 (Cell Signaling Technologies); tubulin, HA-tag, Flag-tag (Sigma-Aldrich); TELO2 (Proteintech); TTI1 (Santa-Cruz Biotechnology); DCAF6, WDR26, eIF3f (Bethyl Labs); and Cul4B (Genetex). Immunoblots were performed as previously described (Dobrikov et al., 2011 (link)). Immunoblot signals were obtained on and quantified using the Li-COR Odyssey FC2 imaging system and Image Studio software. IL-2 (R&D systems) and IFN-γ (Invitrogen) ELISAs were performed using the manufacturer’s instructions.

Brown M.C, & Gromeier M. (2017). MNK Controls mTORC1:Substrate Association Through Regulation of TELO2 Binding with mTORC1. Cell reports, 18(6), 1444-1457.

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Comprehensive Western Blot Analysis

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We used radioimmunoprecipitation assay (RIPA) buffer containing a proteinase and phosphatase inhibitor cocktail (Cell Signaling, #5872, Danvers, MA, USA) for cell lysis for 20 min, followed by DNA shearing by sonication. The following antibodies were used for Western blot experiments: CUL4A, Cell Signaling, Danvers, MA, USA, CUL4B, Sigma HPA011880, Buchs, Switzerland, actin, Santa Cruz I19, Heidleberg, Germany, tubulin, Abcam ab6046 Cambridge, UK, CDT1, Cell Signaling, Danvers, MA, USA, p21 BD Bioscience, San Jose, CA, USA, cleaved caspase-3, Cell Signaling, Danvers, MA, USA, and phospho-histone H3 (Ser10) (pH3), Cell signaling, Danvers, MA, USA. Western blot imaging and signal quantification was performed using Fusion FX7 (Witec AG, Sursee, Switzerland). Uncropped blots with molecular weight markers are provided as Figure S7.

Meerang M., Kreienbühl J., Orlowski V., Müller S.L., Kirschner M.B, & Opitz I. (2020). Importance of Cullin4 Ubiquitin Ligase in Malignant Pleural Mesothelioma. Cancers, 12(11), 3460.

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