Ubiquitin fk2

Manufactured by Enzo Life Sciences

Ubiquitin (FK2) is a monoclonal antibody that recognizes polyubiquitin conjugates. Ubiquitin is a small regulatory protein that marks proteins for degradation by the proteasome. The FK2 antibody can be used to detect the presence of polyubiquitinated proteins in samples.

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8 protocols using ubiquitin fk2

Immunoblotting and Immunofluorescence Antibody Protocol

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Immunoblot analyses were performed using the following antibodies: Akt, Aktp^S473, p70S6K, phospho-p70S6K, 4EBP1, ERK1/2, Atg5, ULK, ERK1/2, phopho-ERK1/2, p38, phospho-p38, phospho-NFκB and phospho-4EBP1 were all purchased from Cell Signaling. FAK (Santa Cruz Biotechnology, Inc.), actin (Cytoskeleton), LC3 (Novus) and FAKp^Y397 (BD Transduction Laboratories) were purchased from the suppliers indicated. Monoclonal anti-HA (16B12) and polyclonal anti-HA (HA.11) were both purchased from Covance. For immunofluorescence studies, LC3 (Millipore), mTOR (Cell Signaling), LAMP-1 and LAMP-2 (Developmental Studies Hybridoma Bank, University of Iowa), p62 (abcam) and ubiquitin (FK2, Enzo Life Sciences) were from the suppliers indicated. Secondary antibodies included goat anti-mouse-Cy3, donkey anti-rabbit Alexa Fluor 488 and goat anti-rat Alexa Fluor 555 and were purchased from Invitrogen. For flow cytometry, TLR4 (Sa15-21; Akashi et al., 2003) was conjugated to biotin and was a kind gift from Jonathan Kagan (Harvard Medical School, Boston, MA). Anti- strepavidin-APC was purchased from Biolegend.

Owen K.A., Meyer C.B., Bouton A.H, & Casanova J.E. (2014). Activation of Focal Adhesion Kinase by Salmonella Suppresses Autophagy via an Akt/mTOR Signaling Pathway and Promotes Bacterial Survival in Macrophages. PLoS Pathogens, 10(6), e1004159.

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Autophagy and Lipid Signaling Pathway Protocols

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The following rabbit antibodies were used: Atg16L1 (#PA1-18296 Thermo Scientific), beclin-1 (#sc-11427 Santa Cruz), and LC3 (#L7543 Sigma, #PM036 MBL). The following mouse antibodies were used: beta-actin (#sc-81178 Santa Cruz), galectin-3 (#556904 BD Pharmingen), and ubiquitin (FK2, #BML-PW8810 Enzo Life Sciences). Pam3CSK4 was purchased from Invivogen. The synthetic SL analog (2,3-dipalmitoyl-2′-sulfate-α-α′-d-trehalose) we used was a kind gift from Drs. Jacques Prandi and Martine Gilleron (Institute of Pharmacology and Structural Biology, Toulouse) [28 (link),29 (link)]. The DIMs were prepared as previously described [30 (link)].

Bah A., Sanicas M., Nigou J., Guilhot C., Astarie-Dequeker C, & Vergne I. (2020). The Lipid Virulence Factors of Mycobacterium tuberculosis Exert Multilayered Control over Autophagy-Related Pathways in Infected Human Macrophages. Cells, 9(3), 666.

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Immunoblotting Quantification and Antibody Validation

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Immunoblotting was performed as described in [28 (link)]. The antibodies used were Ubiquitin (FK2, #BML-PW8810–0100), from Enzo Life Science or Bip (C50B12, #3177), IκBα (N-terminal Antigen, L35A5, #4814) and phospho-elF2α (Ser51, #9721) from Cell Signaling. All the antibodies were used in 1:1000 dilution. As secondary antibodies, anti-mouse/rabbit IRDye800 CW or IRDye680RD (#926–32210, #926–32211, 926–68070, #926–68071) (1:10,000) antibodies were used. Signals were quantified with the LI-COR Odyssey Imager and Image Studio Lite Vers. 5.2. The uncropped original immunoblots are depicted in Supplementary Fig. 1.

del Rio Oliva M, & Basler M. (2022). Valosin-containing protein (VCP/p97) inhibition reduces viral clearance and induces toxicity associated with muscular damage. Cell Death & Disease, 13(12), 1015.

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Antibody Characterization for Cellular Imaging

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The following antibodies/antisera were used in this study. Actin (Cell Signaling Technology); Cer1GAG (mAbP3E9) [24 (link)]; fibrillarin (abcam ab4566), FLAG (Sigma Aldrich); GFP (Wako); PGL-1 (gift from Susan Strome), GFP (Wako); phospho Ser/Thr-Pro (abcam ab9344); ubiquitin (FK2, Enzo Life Sciences). The secondary HRP-conjugated anti-mouse antibodies were from Life Technologies.

Sun B., Kim H., Mello C.C, & Priess J.R. (2023). The CERV protein of Cer1, a C. elegans LTR retrotransposon, is required for nuclear export of viral genomic RNA and can form giant nuclear rods. PLOS Genetics, 19(6), e1010804.

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Autophagy Regulation in Cell Lines

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Diphenyleneiodonium chloride (DPI), Dimethylsulfoxide (DMSO), and Earle's balanced salt solution (EBSS) were purchased from Sigma. Bafilomycin A1 was purchased from Santa cruz. The following rabbit antibodies were used: ULK1 (Cell Signaling), Atg13 (E1Y9V, Cell Signaling), Atg16L1 (Thermo Scientific, MBL), Beclin-1 (Santa Cruz), CD63 (Santa Cruz), LC3 (Sigma, MBL), ndp52 (Abcam). The following mouse antibodies were used: β-actin (Abcam, Santa Cruz), IgG1 K isotype (eBioscience), Galectin-3 (BD Pharmingen), p62 (BD Transduction Laboratories), TLR2 (Invivogen), ubiquitin (FK2, Enzo Life Sciences).

Bah A., Lacarrière C, & Vergne I. (2016). Autophagy-Related Proteins Target Ubiquitin-Free Mycobacterial Compartment to Promote Killing in Macrophages. Frontiers in Cellular and Infection Microbiology, 6, 53.

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Protein Aggregation and Ubiquitination Analysis

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All cell culture and standard reagents were purchased from Sigma. The region of bovine opsin used as an N-glycosylation reporter and epitope tag was as described previously (Johnson et al., 2012 (link)). Anti-TRC40 serum was a gift from Bernhard Dobberstein (ZMBH, Heidelberg, Germany). Commercially available antibodies against the following targets were purchased from the indicated suppliers: BAG6, BAP31, GFP, tubulin and V5 (Abcam), FLAG M2 and calnexin (Sigma), HSP70 (Stressgen), ubiquitin FK2, p23, 20S proteasome (Enzo Life Sciences) and LAMP1 (DHSB, University of Iowa). The antibody against GRASP65 was a gift from Martin Lowe (University of Manchester, UK). ProteoStat® reagent for the detection of protein aggregates was from Enzo. Bortezomib was from Selleck Chemicals, leupeptin and pepstatin A were from BIOMOL. RFP in pcDNA3.1⁺ was a gift from Viki Allan (Manchester, UK). siRNA duplexes for knockdowns were from Qiagen, and they targeted sequences that were identified previously (Winnefeld et al., 2006 (link)): SGTA target sequence, 5′-TTTGAAGCTGCCGTGCATT-3′; BAG6 target sequence, 5′-CAGCTCCGGTCTGATATACAA-3′.

Wunderley L., Leznicki P., Payapilly A, & High S. (2014). SGTA regulates the cytosolic quality control of hydrophobic substrates. Journal of Cell Science, 127(21), 4728-4739.

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Organelle Protein Regulation Assay

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MG132 (Enzo Biochem), NMS873 (Selleck Chemicals), bortezomib, bafilomycin A1, and MLN4924 (EMD Millipore) were dissolved in DMSO, stored at −80°C, and used at the indicated concentrations. DMSO at 0.1% served as the vehicle control.
Antibodies used in this study were SLC25A46 (Proteintech); HA (HA.11; Biolegend); MFN1 (ab57602) and histidine (HIS.H8; Abcam); LC3A/B (Cell Signaling); MFN2 (D1E9; Cell Signaling); MTCH2 (Abgent); MIC60/IMMT (Abgent and Proteintech); MID51 (Proteintech); Ubiquitin (FK2, Enzo Biochem); Mortalin (University of California, Davis/National Institutes of Health NeuroMab Facility); MIC19/CHCHD3 (Everest Biotech); MIC27/Apool (Assay BioTech); OPA1, P97/VCP, and TIMM23 (BD Biosciences); TOMM20 (FL-145), LRP130 (H-300), and glyceraldehyde-3-phosphate dehydrogenase (FL-335; Santa Cruz Biotechnology); MULAN (HPA017681) and Flag (M2) (Sigma-Aldrich); MARCH5 (provided by Richard Youle); myc (9E10; Developmental Studies Hybridoma Bank, University of Iowa); and Cullin 4a (PA5-29857; Thermo Fisher Scientific). PREP, GFP, TOMM40, YME1L, and PNPase polyclonal antibodies were raised against recombinant proteins (Pacific Immunology).

Steffen J., Vashisht A.A., Wan J., Jen J.C., Claypool S.M., Wohlschlegel J.A, & Koehler C.M. (2017). Rapid degradation of mutant SLC25A46 by the ubiquitin-proteasome system results in MFN1/2-mediated hyperfusion of mitochondria. Molecular Biology of the Cell, 28(5), 600-612.

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Recombinant Nanobodies for VacA/VacB

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Recombinant nanobodies with the Fc portion of rabbit IgG, which recognize specifically VacA or VacB were previously characterized (Bosmani et al., 2020) . The other following antibodies were used: pan-vacuolin (Dr. M. Maniak (Jenne et al., 1998) ), VatA (Dr. M. Maniak (Jenne et al., 1998) ), p80 (purchased from the Geneva Antibody Facility), cathepsin D (Dr. J. Garin (Journet et al., 1999) ), ubiquitin FK2 (Enzo Life Sciences), and GFP (pAb from MBL Intl., mAb from Abmart). Goat antimouse or anti-rabbit IgG coupled to AlexaFluor 488, AlexaFluor 594, AlexaFluor 647 (Invitrogen) or to HRP (Brunschwig) were used as secondary antibodies.
After SDS-PAGE separation and transfer onto nitrocellulose membranes (Protran), immunodetection was performed as previously described (Schwarz et al., 2000) but with ECL Prime Blocking Reagent (Amersham Biosciences) instead of non-fat dry milk. Detection was performed with ECL Plus (Amersham Biosciences) using a Fusion Fx device (Vilber Lourmat). Quantification of band intensity was performed with ImageJ.
For immunofluorescence, infected D. discoideum cells were fixed with ultra-cold methanol (MeOH) at the indicated time points and immunostained as previously described (Hagedorn et al., 2006) . Images were recorded with a Leica SP8 confocal microscope using a 63×1.4 NA oil immersion objectives.

Bosmani C., Perret A., Leuba F., Guého A., Hanna N., & Soldati T. (2021). Disruption of vacuolin microdomains in the host Dictyostelium discoideum increases resistance to Mycobacterium marinum-induced membrane damage and infection.

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