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Cullin3

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Cullin3 is a protein that is a core component of E3 ubiquitin ligase complexes. It functions to facilitate the ubiquitination and subsequent degradation of target proteins.

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Lab products found in correlation

Cullin4b, by Proteintech (2 mentions) Anti flag antibody, by Merck Group (1 mentions) Nedd4 1, by Cell Signaling Technology (1 mentions) Anti spop antibody, by Proteintech (1 mentions) Epoxomicin, by Merck Group (1 mentions) Z ietd fmk, by R&D Systems (1 mentions) Nedd8, by Cell Signaling Technology (1 mentions) Z vad fmk, by R&D Systems (1 mentions) Procaspase 3, by Cell Signaling Technology (1 mentions) Recombinant rat tnf α, by Thermo Fisher Scientific (1 mentions) Pro caspase 7, by Cell Signaling Technology (1 mentions) Phospho iκbα, by Cell Signaling Technology (1 mentions) Cleaved caspase 3, by Cell Signaling Technology (1 mentions) β actin, by Cell Signaling Technology (1 mentions) Necrostatin 1, by Merck Group (1 mentions) Ubiquitin, by Cell Signaling Technology (1 mentions) Xbp1s, by Cell Signaling Technology (1 mentions) β actin hrp, by Abcam (1 mentions) Imagequant tl software, by GE Healthcare (1 mentions) Cullin1, by Cell Signaling Technology (1 mentions)

5 protocols using cullin3

1

Protein Immunoprecipitation and Analysis

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IFN-γ (cat#: Z02915) was purchased from GenScipt (Piscataway, NJ). Antibodies to SNX1 (cat#: 611482) and SNX2 (cat#: 611308) were from BD Bioscience (San Jose, CA). Antibody to SNX5 (cat#: ab180520) was from Abcam (Cambridge, MA). Antibodies to SNX6 (cat#: sc-8679) and tubulin (cat#: sc-398103) were from Santa Cruz Biotechnology (Santa Cruz, California). Antibodies to PD-L1 (cat#: 13684), NEDD4-1 (cat#: 2740), Cullin3 (cat#: 2759), and Myc (cat#: 2778) were from Cell Signaling (Danvers, MA). Anti-SPOP antibody (cat#: 16750-1-AP) was from Proteintech (Rosemont, IL). Anti-Flag antibody (cat#: F3165) was from Sigma-Aldrich (St. Louis, MO). Secondary antibodies were obtained from Jackson Immuno Research Laboratories (West Grove, PA). Human recombinant SNX6 protein (GST tag) and recombinant Cullin3 protein (Flag tag) were purchased from OriGene (Rockville, MD).
Ghosh C., Xing Y., Li S., Hoyle R.G., Sun M., Li J, & Sun Y. (2021). Sorting Nexin 6 Interacts with Cullin3 and Regulates Programmed Death-ligand 1 Expression. FEBS letters, 595(20), 2558-2569.
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2

Pevonedistat Synthesis and Reagent Details

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Pevonedistat was synthesized by Millennium Pharmaceuticals, Inc. The following reagents were purchased from their respective companies: recombinant rat TNF-α (Peprotech, Rocky Hill, NJ, USA); caspase inhibitors Z-VAD-FMK and Z-IETD-FMK (R&D Systems, Minneapolis, MN, USA); Necrostatin-1 (Sigma-Aldrich, St. Louis, MO, USA); and Epoxomicin (Sigma-Aldrich). Antisera were purchased from the following companies: β-Actin, cleaved caspase-3, cleaved caspase-8 (p18), cFLIP, cullin-3, IκBα, phospho-IκBα, NEDD8, PARP, pro-caspase-3, pro-caspase-6, pro-caspase-7, pro-caspase-8 (p10), pro-caspase-9 (Cell Signaling, Danvers, MA, USA); MLKL (Millipore, Billerica, MA, USA); CDT1 (Santa Cruz Biotechnology, Dallas, TX, USA); and BID (eBioscience, San Diego, CA, USA). Complete antisera details are provided in Supplementary Information.
Wolenski F.S., Fisher C.D., Sano T., Wyllie S.D., Cicia L.A., Gallacher M.J., Baker R.A., Kirby P.J, & Senn J.J. (2015). The NAE inhibitor pevonedistat (MLN4924) synergizes with TNF-α to activate apoptosis. Cell Death Discovery, 1, 15034-.
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3

Quantitative Western Blotting Technique

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Western blot was performed using 10–30 μg of total protein lysates or 20 μg of nuclear extracts. The following antibodies were used in this study: XBP1s (Cell Signaling, 83418), CHOP (Cell Signaling, 2895), PARP (Cell Signaling, 9532), ubiquitin (Cell Signaling, 3933), Cullin1 (Cell Signaling, 4995), Cullin3 (Cell Signaling, 2759), Cullin4B (Proteintech, 12916-1-AP), SPOP (Proteintech, 16750-1-AP), Flag (Sigma, F1804 and F7425), Myc (Cell Signaling, 2278 and 2276), HA (Sigma, H3663; and Cell Signaling, 3724), βActin-HRP (Abcam, ab49900), and HSP90 (Santa Cruz Biotechnology, sc7947). Secondary antibodies—donkey antirabbit or mouse IgG HRP (Jackson ImmunoResearch)—were used at 1:6000 dilution. Western blot membranes were developed using the Clarity Western ECL substrate (Bio-Rad), and signal was detected with an ImageQuant LAS4000 luminescent imager (General Electric). Quantification was performed using ImageQuantTL software (General Electric).
Sun S., Kelekar S., Kliewer S.A, & Mangelsdorf D.J. (2019). The orphan nuclear receptor SHP regulates ER stress response by inhibiting XBP1s degradation. Genes & Development, 33(15-16), 1083-1094.
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4

Cullin-RING Ubiquitin Ligase Toolkit

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Antibodies specific to C-PARP (Cell Signaling Technology, 5625), Cullin1 (Abcam, 75817), Cullin2 (Abcam, 166917), Cullin3 (Cell Signaling Technology, 2759), Cullin4A (Cell Signaling Technology, 2699), Cullin4B (Proteintech, 12916-1-AP), Cullin5 (Abcam, 184177), NAE (Cell Signaling Technology, 14321), NOXA (Cell Signaling Technology, 14766), P27 (Cell Signaling Technology, 3686), RBX1 (Abcam, 133565), UBA3 (Abcam, 124726), UBE2F (Abcam, 12932), and UBE2M (Abcam, 109507) were purchased commercially.
Zhou L., Zhu J., Chen W., Jiang Y., Hu T., Wang Y., Ye X., Zhan M., Ji C., Xu Z., Wang X., Gu Y, & Jia L. (2020). Induction of NEDD8-conjugating enzyme E2 UBE2F by platinum protects lung cancer cells from apoptosis and confers to platinum-insensitivity. Cell Death & Disease, 11(11), 975.
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5

Cullin Protein Expression Analysis

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Western blotting was performed as described previously22 (link). Treated cells were lysed by RIPA buffer supplemented with protease inhibitor. The expression level of indicated proteins was examined by immunoblotting analysis and GAPDH was used as the loading control. Antibodies were purchased commercially: cullin 1 (sc-17775, 1:500 dilution) and cullin 2 (sc-166506, 1:500 dilution) from Santa Cruz Biotech (Santa Cruz, CA); cullin 4A (PA5-14542, 1:1000 dilution), cullin 4B (PA5-50647, 1:1000 dilution), and DCN3 (DCUN1D3, PA5-44000, 1:1000 dilution) from Thermo Fisher Scientific (Wayne, MI); cullin 3 (2759, 1:1000), NRF2 (12721, 1:1000 dilution), HO-1 (70081, 1:1000 dilution), and Bim (2819, 1:1000 dilution) from Cell Signaling Technology (Boston, MA); DCN1 (GWB-E3D700, 1:1000 dilution) from GenWay Biotech (San Diego, CA); and cullin 5 (A302-173A, 1:500 dilution) from Bethyl Labs (Montgomery, TX). Results are representative of two independent experiments.
Zhou H., Lu J., Chinnaswamy K., Stuckey J.A., Liu L., McEachern D., Yang C.Y., Bernard D., Shen H., Rui L., Sun Y, & Wang S. (2021). Selective inhibition of cullin 3 neddylation through covalent targeting DCN1 protects mice from acetaminophen-induced liver toxicity. Nature Communications, 12, 2621.
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