Anti ubiquitin fk2

Manufactured by Enzo Life Sciences

Sourced in United States, United Kingdom

Anti-ubiquitin (FK2) is a monoclonal antibody produced by Enzo Life Sciences. It recognizes polyubiquitinated conjugates and can be used to detect ubiquitinated proteins.

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Ubiquitin (18 citations) Anti-ubiquitin (17 citations) Ubiquitin (FK2, (8 citations) Mouse anti-Ubiquitin FK2 (3 citations) Anti-ubiquitin clone FK2 (3 citations) Anti-ubiquitin antibody (clone FK2) (2 citations) Anti-ubiquitin clone FK2 and anti-LAMP1 clone Ly1C6 (2 citations)

9 protocols using anti ubiquitin fk2

Tandem Affinity Purification of Ubiquitinated Proteins

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TAP was performed as described previously, with minor modifications (Gloeckner et al., 2007 (link); Peters et al., 2013 (link)). RAW247.1 macrophages were infected overnight with 2 p.f.u. per cell and subsequently lysed in PBS supplemented with 0.5 % NP-40. Lysates were incubated with streptavidin beads (Thermo Scientific) for 2 h at 4 °C. Beads were washed three times and eluted in PBS supplemented with biotin (Thermo Scientific) for 1 h at 4 °C. Eluates were incubated with anti-FLAG M2 agarose beads (Sigma) and eluted in PBS supplemented with FLAG peptide (Sigma) in a similar manner. Final eluates were concentrated using 3 kDa Amicon filters (Millipore). Proteins retained on streptavidin and FLAG beads, as well as final eluates, were resolved in Novex 4–12 % Bis-Tris protein gels (Invitrogen) and analysed by silver staining (Invitrogen), or immunoblotting with anti-FLAG (Sigma-Aldrich) or anti-ubiquitin (FK2; Enzo Life Sciences) antibodies.

Maluquer de Motes C., Schiffner T., Sumner R.P, & Smith G.L. (2014). Vaccinia virus virulence factor N1 can be ubiquitylated on multiple lysine residues. The Journal of General Virology, 95(Pt 9), 2038-2049.

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Ubiquitination of GluA1 receptors

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Equal amounts (2 mg) of lysate proteins were collected from hippocampal slices treated with DMSO or G1+DHPG in the presence of MG132. Samples were immunoprecipitated with protein A/G agarose beads (Thermo Fisher Scientific) previously coated with 4 µg of either GluA1 antibody (AB1504) or IgG (sc-2027). Anti-ubiquitin (Fk2; Enzo Life Sciences) and anti-GluA1 (AB1504 and sc-13152) antibodies were used for immunoblotting.

Briz V., Liu Y., Zhu G., Bi X, & Baudry M. (2015). A novel form of synaptic plasticity in field CA3 of hippocampus requires GPER1 activation and BDNF release. The Journal of Cell Biology, 210(7), 1225-1237.

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Molecular Tools for Autophagy Research

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HA-NEDD4^WT (Addgene, 27002), and HA-NEDD4^C867A (Addgene, 26999) were gifts from Dr. Joan Massague.⁵⁸ (link) MYC-ULK1^WT (Addgene, 27629), MYC-ULK1^K46I (Addgene, 27630), and MYC-ULK1^S4A (Addgene, 27631) were gifts from Dr. Reuben Shaw.⁵⁹ (link) FLAG-ubiquitin WT and related mutants (K6WT, K11WT K27WT, K29WT, K33WT, K48WT, K63WT and KallR) were provided by the MRC Protein Phosphorylation and Ubiquitylation Unit, UK. YFP-Atg8-family proteins encoding plasmids were a kind gift from Dr. Felix Randow (MRC Laboratory of Molecular Biology, UK). MYC-BECN1 plasmid (Biocat GmbH, MR207162-OR), MG132 (Merck Chemicals GmbH, 474791), Torin1 (Tocris Bioscience, 4247), cyclohexamide (Sigma-Aldrich, C1988) and choloroquine (Sigma-Aldrich, C6628) were purchased as indicated. Antibodies used in this study include: anti-ACTB (Sigma-Aldrich, A2228), anti-LC3 (MBL International, PM036), anti-human NEDD4 (Cell Signaling Technology, 3607), anti-mouse NEDD4 (BD Biosciences, 611481), anti-K48 ubiquitin (Merck Millipore, 05–1037), anti-K63 ubiquitin (Enzo Life Sciences, HWA4C4), anti-ubiquitin (FK2) (Enzo Life Sciences, BML-PW8810), rabbit anti-BECN1 (Cell Signaling Technology, 3495), mouse anti-BECN1 (Cell Signaling Technology, 4122), anti-ULK1 (Cell Signaling Technology, 8054), anti-LC3B (Sigma-Aldrich, L7543), anti-LAMP2 (Santa Cruz Biotechnology, SC-18822).

Pei G., Buijze H., Liu H., Moura-Alves P., Goosmann C., Brinkmann V., Kawabe H., Dorhoi A, & Kaufmann S.H. (2017). The E3 ubiquitin ligase NEDD4 enhances killing of membrane-perturbing intracellular bacteria by promoting autophagy. Autophagy, 13(12), 2041-2055.

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Detailed Immunoblotting Antibody Protocol

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The following antibodies were used according to manufacturer’s and/or published suggestions for immunoblotting: anti-β-Actin (Abcam), anti-Biotin (Cell Signaling), Streptavidin-AF647 (Invitrogen), anti-Arc (Gift from P. Worley, Johns Hopkins, verified against knockout), anti-Fos (Cell Signaling), anti-Npas4 (Gift from Y. Lin, MIT, verified against knockout), anti-PSD-95 (Pierce), anti-UBE3A (Sigma, verified against knockout), anti-Ubiquitin (FK2, Enzo), anti-S6 ribosomal subunit (Cell Signaling), anti-Transferrin receptor (Sigma), anti-β2 proteasome (Enzo), anti-α1–7 proteasome (Enzo). Standard secondary antibodies were purchased from Cell Signaling. We attempted to use antibodies that were verified by knockout controls in either our study, or by other groups. We only used antibodies that provided a signal at the appropriate molecular weight, and where minimal nonspecific bands were observed.

Ramachandran K.V., Fu J.M., Schaffer T.B., Na C.H., Delannoy M, & Margolis S.S. (2018). Activity-dependent degradation of the nascentome by the neuronal membrane proteasome. Molecular cell, 71(1), 169-177.e6.

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Antibody Characterization for Autophagy Study

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Antibodies used for the study were: anti-Atg8 (LSBio, LS-B4021),
anti-GFP (Molecular Probes, A6455), anti-phospho-Ser (Santa Cruz, sc-81514),
anti-phospho-Threnonine (Cell Signaling, 9381), anti-Phospho-PKA substrate
(Cell Signaling, 9624), anti-Flag (Sigma, F3165), anti-HA
(Covance/BioLegend, MMS-101P), anti-CDK8 (Santa Cruz, sc-13155 ), anti-LC3B
(Cell Signaling, 2775), anti-human CPSF6 (Santa Cruz, sc-100692),
anti-ubiquitin FK2 (Enzo life Science, BML-PW8810-0100), and anti-Tubulin
(Sigma, T5168). Anti-Atg1 was a gift of Dr. Jun Hee Lee (Kim et al., 2013 (link)). Anti-CLK2 was produced by
cloning via PCR a 1.3 kb fragment encoding the catalytic domain of human
CLK2 from a full-length cDNA in frame into the BamHI site in the pMAL-C2
vector (New England Biolabs). It was expressed in E. colias a fusion protein with the Maltose Binding Protein. Following purification
on an amylose column, the recombinant protein was injected into two rats for
antibody production.

Tang H.W., Hu Y., Chen C.L., Xia B., Zirin J., Yuan M., Asara J.M., Rabinow L, & Perrimon N. (2018). The TORC1-regulated CPA complex rewires an RNA processing network to drive autophagy and metabolic reprogramming. Cell metabolism, 27(5), 1040-1054.e8.

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Malignant Peripheral Nerve Sheath Tumor Cell Lines

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The human MPNST cell lines sNF96.2 and sNF02.2 were purchased from ATCC (ATCC, Manassas, VA), while ST8814 and STS26T cells were kind gifts from Dr. Yang Jilong (Tianjin Medical University, China) and Dr. Nancy (Cincinnati Children’s Hospital Medical Center, USA). All cells were cultured in Dulbecco’s modified Eagle’s medium (Gibco) supplemented with 10% FBS, and they were maintained at 37 °C in a humidified atmosphere with 5% CO₂.
The following antibodies were used in the experiments: anti-E-cad, anti-N-cad, anti-Vimentin and anti-GAPDH antibodies from Cell Signaling Technology (Beverly, MA, USA); anti-MSI2 and anti-CAV1 antibodies from Abcam (Cambridge, MA, USA),anti-ubiquitin (FK2) from Enzo Life Sciences(New York, NY, USA).

Yang K., Du J., Shi D., Ji F., Ji Y., Pan J., Lv F., Zhang Y, & Zhang J. (2020). Knockdown of MSI2 inhibits metastasis by interacting with caveolin-1 and inhibiting its ubiquitylation in human NF1-MPNST cells. Cell Death & Disease, 11(6), 489.

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Antibody Validation for Western Blotting

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The following primary antibodies were used for Western blotting: anti-GFP (Roche Applied Science, monoclonal and in-house polyclonal antibody raised against GFP(2–238)); anti-ubiquitin FK2 (Enzo) and anti-HA tag (Cell Signaling and Bethyl Laboratories); anti-His tag (Cell Signaling); anti-DCNL1 clone 3D7 (Sigma); anti-β-actin (Cell Signaling); anti-CUL1 (Invitrogen); anti-CUL2 (Invitrogen); anti-CUL4B (Sigma); anti-Elongin-C (BioLegend); anti-RBX1 (ThermoFisher Scientific); anti-CAND1, anti-IκBα, and anti-phospho(S536)–p65 (Cell Signaling); anti-CSN5 (Abcam); anti-Hif1α (R&D Systems); anti-RBX2 (Abcam); anti-CSN3 (Bethyl Lab); anti-CSN7B (Epitomics); anti-CSN8 (Abcam); and anti-histone H2A (Abcam). Polyclonal antibodies against CUL3, CUL4A, CUL5, HHARI, and TRIAD1 have been described previously (28 (link)).

Kelsall I.R., Kristariyanto Y.A., Knebel A., Wood N.T., Kulathu Y, & Alpi A.F. (2018). Coupled monoubiquitylation of the co-E3 ligase DCNL1 by Ariadne-RBR E3 ubiquitin ligases promotes cullin-RING ligase complex remodeling. The Journal of Biological Chemistry, 294(8), 2651-2664.

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Comprehensive Western Blot Analysis

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Western blotting was performed as previously described (van den Boom et al., 2013 (link)). The following primary antibodies were used in this study: anti-KDM2B (09-864, Merck), anti-USP7 (A300-033A, Bethyl Laboratories), anti-TRIM27 (18791, IBL), anti-RING1A (ab180170, Abcam), anti-RING1B (ab181140, Abcam), anti-PCGF1 (ab183499, Abcam), anti-STAT5 (sc-835-G, Santa Cruz Biotechnology), anti-TP53 (sc-216, Santa Cruz Biotechnology), anti-MDM2 (sc-813, Santa Cruz Biotechnology), anti-GFP (sc-9996, Santa Cruz Biotechnology), anti-GFP (ab290, Abcam), anti-Ubiquitin (FK2, Enzo Life Sciences), anti-H2AK119ub (D27C4, Cell Signaling), and anti-b-Actin (C4, Santa Cruz Biotechnology). Secondary antibodies used included either goat anti-mouse IRDye 800 or goat anti-rabbit IgG (H+L) Alexa Fluor 680 (Invitrogen) for imaging using an Odyssey CLx Imaging System (Li-Cor Biosciences), or goat anti-rabbit immunoglobulins/HRP (Agilent-Dako), and rabbit anti-mouse immunoglobulins/HRP (Agilent-Dako) for imaging using a ChemiDoc XRS+ System (Biorad).

Maat H., Atsma T.J., Hogeling S.M., Rodríguez López A., Jaques J., Olthuis M., de Vries M.P., Gravesteijn C., Brouwers-Vos A.Z., van der Meer N., Datema S., Salzbrunn J., Huls G., Baas R., Martens J.H., van den Boom V, & Schuringa J.J. (2021). The USP7-TRIM27 axis mediates non-canonical PRC1.1 function and is a druggable target in leukemia. iScience, 24(5), 102435.

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Ubiquitination and SUMOylation Assay

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Etoposide and 2′,3′,4′-trihydroxy-flavone (2-D08) were purchased from Sigma-Aldrich (Dorset, UK); PR-619 was obtained from Tocris Biosciences (Bristol UK); {(1R,2R,3S,4R)-2,3-dihydroxy-4-[(2-{3-[(trifluoromethyl)sulfanyl]phenyl}pyrazolo[1,5-a]pyrimidin-7-yl)amino]cyclopentyl}methyl sulfamate (MLN7243) was obtained from Active Biochem Ltd. (Hong Kong); and {(1R,2S,4R)-4-[(5-{[1-(3-bromobenzyl)-1H-pyrazol-3-yl]carbonyl}-4-pyrimidinyl)amino]-2-hydroxycyclopentyl}methyl sulfamate (ML-792) was obtained from Bioquote (York, UK). Rabbit anti-TOP2A (4566) and anti-TOP2B (4555) antibodies were raised in-house to the C-terminal domains of the respective proteins (Atwal et al., 2019 (link)). Anti-ubiquitin FK2 (anti-mono and poly-ubiquitinated conjugates, K²⁹, K⁴⁸, or K⁶³ linked) was obtained from Enzo (Exeter, UK), and anti-SUMO2/3 (Ab81371) was from Abcam (Cambridge UK).

Cowell I.G., Ling E.M., Swan R.L., Brooks M.L, & Austin C.A. (2019). The Deubiquitinating Enzyme Inhibitor PR-619 is a Potent DNA Topoisomerase II Poison. Molecular Pharmacology, 96(5), 562-572.

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