Guaranteed quality will yield reproducible, quantitative results and there are no contaminations with heavy or light chains. 1B). We also examined the plausible compensation by NBR1 (Neighbor of Braca 1 gene), a redundant autophagic receptor that can effectively substitute for p62 in its absence (59) (supplemental Figs. However, most of the proteins identified were novel IB-interactors. Scale bar, 10 m. To determine the physiological significance of IB-NUP153 interaction and their concurrent aggregation, we monitored their interaction through co-immunoprecipitation (Co-IP) using HEK293T cells. Fisher Scientific - Proteintech GFP VHH conjugated to Alexa Fluor 647. Cells were fixed and stained with anti-p65/anti-rabbit-Alexa-488 IgGs (green) and anti-IB/anti-mouse Alexa-647-conjugated IgGs (magenta) and DAPI. (2013), Lamin aggregation is an early sensor of porphyria-induced liver injury, Currais A., Fischer W., Maher P., and Schubert D. (2017), Intraneuronal protein aggregation as a trigger for inflammation and neurodegeneration in the aging brain, Nivon M., Fort L., Muller P., Richet E., Simon S., Guey B., Fournier M., Arrigo A. P., Hetz C., Atkin J. D., and Kretz-Remy C. (2016), NFkappaB is a central regulator of protein quality control in response to protein aggregation stresses via autophagy modulation, Regulation of the NF-kappa B/rel transcription factor and I kappa B inhibitor system, NF-kappaB and STAT3 - key players in liver inflammation and cancer, Azimifar S. B., Nagaraj N., Cox J., and Mann M. (2014), Cell-type-resolved quantitative proteomics of murine liver, Mechanism for biphasic rel A. NF-kappaB1 nuclear translocation in tumor necrosis factor alpha-stimulated hepatocytes, Rao P., Hayden M. S., Long M., Scott M. L., West A. P., Zhang D., Oeckinghaus A., Lynch C., Hoffmann A., Baltimore D., and Ghosh S. (2010), IkappaBbeta acts to inhibit and activate gene expression during the inflammatory response, Winiewski J. R., Vildhede A., Norn A., and Artursson P. (2016), In-depth quantitative analysis and comparison of the human hepatocyte and hepatoma cell line HepG2 proteomes, Beg A. Imprint (Impressum) Such an apparent IB-loss was also resistant to various UPD, calpain and ALD inhibitors (Fig. To our knowledge, this is the first evidence that NF-B is also activated through IB-sequestration into insoluble aggregates. HEK293T cells were transfected with C1-GFP control vector, or C1-GFP-IB vector, or a mock control for 40 h. Co-IP of cytosolic fractions (CER) and nuclear fractions (NER) with GFP-trap followed by Nup153 and IB IB analyses, with actin as the loading control, and GAPDH and HistoneH3 (H3) as fractionation markers. B, Primary hepatocytes from WT (p62WT) and p62 knockout (p62KO) mice were treated with ZnPP (10 M) for the indicated times. Nuclear fractions of cells were prepared using NE-PER Nuclear and Cytoplasmic Extraction Reagents (ThermoFisher, Grand Island, NY). AbbreviationsThe abbreviations used are: National Library of Medicine Cells were washed 3 times with PBS and then lysed in RIPA buffer supplemented with 2% SDS and boiled for 1 h to reverse formaldehyde-cross-linking. As a library, NLM provides access to scientific literature. Confocal image of HeLa cells transiently transfected with Tom70-eGFP and immunostained with GFP-Booster Alexa Fluor 488 (green). Cells were allowed to attach for 4 to 6 h and then overlaid with Matrigel (Corning, Oneonta, NY). Copyright 2002-2022 Proteintech Group, Inc. All rights reserved. ZnPP-elicited IB-loss is due to its sequestration into insoluble cellular aggregates. Negative control staining with no antibody or staining with mouse IgG1 isotype controls (adjusted to the same final concentration as that of IB-antibody) was also performed. C, Box plots of the proteins with long intrinsic disordered regions (regions with >30 consecutive disordered residues) and relative protein hydrophobicity. Because RanBP2 also strongly binds RanGDP, conceivably a RanBP2-RanGDP-Nup153-IB-SUMO complex is involved in IB-nuclear import, a possibility consistent with our RanBP2-knockdown analyses (Fig. ), GM097057 (D.H.K. None of the 10 proteins that interacted with IB and concurrently aggregated upon ZnPP-treatment are well-established stable IB-interactors. Cell lysates were used for IB analyses. A, HEK293T cells were transfected with pCMV4-3HA-IB for 40 h, and then treated with ZnPP (10 M) for 4 h. Cells were then subjected to sequential extraction. pCMV-3HA-IB and pCMV-3HA-IB-S32A/S36A were gifts from Dr. Warner Greene (plasmids #21985, #24143; Addgene, Cambridge, MA). Protect from light. Of these 10 proteins, the nucleoporins Nup153 and Nup358/RanBP2 were identified through RNA-interference, as mediators of IB-nuclear import. Functional enrichments are annotated with color-lined boxes with the false discovery rates (FDR) included. And their Chromobodies allow you to analyze endogenous proteins in living cells in real time. (2015), The ubiquitin kinase PINK1 recruits autophagy receptors to induce mitophagy, Micro-scale photofluorometric determination of free erythrocyte pophyrin (protoporphyrin IX), Ku N. O., Toivola D. M., Zhou Q., Tao G. Z., Zhong B., and Omary M. B. ChromoTek GFP-Trap is the most cited Nanobody-based product of the year 2022. pcDNA6-HA-rHRI, C1-Emerald-IB, pcDNA6-p62-myc and pcDNA3-NBR1 were constructed by us. In parallel with IB-loss, p62 formed aggregates and possibly cross-linked species, with a concurrent decline in the monomeric species ( 62 kDa; Ctrl), evident at later (8 h) rather than earlier (24 h) time points. Proteintech ChromoTek GFP-Booster ATTO647N 10 L Proteintech ChromoTek However, the precise mechanism that links protein aggregation to NF-B-activation and inflammatory response remains unclear. BAR analyses following ZnPP-treatment were employed in the main analyses (Figs. (2015), Ambient Light Promotes Selective Subcellular Proteotoxicity after Endogenous and Exogenous Porphyrinogenic Stress, Elenbaas J. S., Maitra D., Liu Y., Lentz S. I., Nelson B., Hoenerhoff M. J., Shavit J. We thus examined their interdependence in ZnPP-treated p62 WT and p62 null mouse (p62KO) hepatocytes or MEF cells (Figs. Total RNA was extracted using RNeasy Mini Kit (Qiagen, Germantown, MD) according to the manufacturers' instructions. This IB-loss was reversed upon inclusion of hemin, but not that of the proteasomal inhibitor MG-132, or the dual autophagic-lysosomal degradation (ALD) inhibitors 3-methyladenine (3MA) + NH4Cl. (2016), A precursor-inducible zebrafish model of acute protoporphyria with hepatic protein aggregation and multiorganelle stress, Klement J. F., Rice N. R., Car B. D., Abbondanzo S. J., Powers G. D., Bhatt P. H., Chen C. H., Rosen C. A., and Stewart C. L. (1996), IkappaBalpha deficiency results in a sustained NF-kappaB response and severe widespread dermatitis in mice, Tergaonkar V., Correa R. G., Ikawa M., and Verma I. M. (2005), Distinct roles of IkappaB proteins in regulating constitutive NF-kappaB activity, Sequestration of cellular interacting partners by protein aggregates: implication in a loss-of-function pathology, Lahiri P., Schmidt V., Smole C., Kufferath I., Denk H., Strnad P., Rulicke T., Frohlich L. F., and Zatloukal K. (2016), p62/Sequestosome-1 Is Indispensable for Maturation and Stabilization of Mallory-Denk Bodies, Olzscha H., Schermann S. M., Woerner A. C., Pinkert S., Hecht M. H., Tartaglia G. G., Vendruscolo M., Hayer-Hartl M., Hartl F. U., and Vabulas R. M. (2011), Amyloid-like aggregates sequester numerous metastable proteins with essential cellular functions, Molecular mechanisms of system control of NF-kappaB signaling by IkappaBalpha, Fagerlund R., Kinnunen L., Kohler M., Julkunen I., and Melen K. (2005), NF-{kappa}B is transported into the nucleus by importin {alpha}3 and importin {alpha}4, Lu M., Zak J., Chen S., Sanchez-Pulido L., Severson D. T., Endicott J., Ponting C. P., Schofield C. J., and Lu X. Specificity: 1x and 3x DYKDDDDKsequence (1x and 3xFlag-tag)at the N-terminus, C-terminus, or internal site of the fusion protein.Applications: IP/Co-IP, MS, protein purification, on-bead enzyme assays, ChIP/RIP analysisFormats: Agarose. The GFP-Booster stabilizes, enhances, and reactivates the signal of GFP-fusion proteins. document.getElementById('cloak4664149d03117902069574ef3f48ed77').innerHTML = ''; ChromoTek GmbH Privacy Policy Use your existing GFP/RFP fusion protein for biochemical studies and super-resolution microscopy. ChromoTek offers innovative research reagents based oncamelid antibodies, which aredevoid of light chains and have a single antigen recognizing domain. S5B). 4B, supplemental Tables S7S10). 8B). Chat with a team member. S6B), suggesting that NF-B p65 no longer binds to IB once the latter aggregates upon ZnPP-treatment. orders@lubio.ch, Monday - Friday This additional paracrine cytokine stimulus could further potentiate the PPIX-elicited NF-B-signaling activation within the hepatocyte, further aggravating the extent of EPP and XLPP liver injury. Although different aggregates vary with each tissue source and in predominant protein composition, they contain common components such as p62/Sequestosome-1, ubiquitin, chaperones and proteasome constituents, which are often misfolded, highly insoluble, and cross-linked (1, 2). A, HEK293T cells were transfected with pCMV4-3HA-IB, or co-transfected with either pcDNA6-p62-Myc or pcDNA6-Myc empty vector for 48 h. Cell lysates were used for IB analyses with actin as the loading control. B, The same extracts from (A) were immunoblotted using three different antibodies each targeting a different region of the same HA-IB protein: Anti-HA-tag, rabbit mAb targeting IB N terminus (N), and rabbit mAb targeting IB-C terminus (C). For super-shift EMSA, the binding mixture (in the absence of the biotin-labeled probes) was first incubated with p65 antibody for 20 min on ice, and then the biotin-labeled probes were added and further incubated at room temperature for another 30 min before gel loading. Specificity on Phospho-MK2 (Thr334) was not tested.Applications: IP/Co-IP, MS, on-bead enzyme assaysFormats: Agarose, Specificity: mNeonGreen, a bright monomeric yellow-green fluorescent protein derived from the lanceletBranchiostoma lanceolatum, and othersApplications: IP/Co-IP, MS, on-bead enzyme assays, ChIP/RIP analysisFormats: Agarose, magnetic agarose, iST kit, Specificity: Myc-tag motif EQKLISEEDL at the N-terminus, C-terminus, or internal site of the fusion protein. The resulting pellet was then solubilized in half the volume (150 l) of cell lysis buffer with sonication in urea/CHAPS buffer containing 8 M urea, 2 M thiourea, 4% CHAPS, 20 mM Tris-base, and 30 mM DTT and supplemented with protease/phosphatase inhibitor mixture. 7A). Mallory-Denk-bodies, IB, , IB, , NF-B, p62, erythropoietic protoporphyria, X-linked protoporphyria, liver inflammation, NMPP, PPIX, ZnPP, proteomics., affinity proteomics, hepatotoxicity, inflammation, inflammatory response, knockouts*, label-free quantification, liver disease, mass spectrometry, protein-protein interactions, immunoaffinity, IkBa, NF-kB, protein aggregation, ZnPPIX, {"type":"entrez-nucleotide","attrs":{"text":"B40911","term_id":"2545163"}}, {"type":"entrez-protein","attrs":{"text":"P49790","term_id":"206729891"}}, {"type":"entrez-protein","attrs":{"text":"P49792","term_id":"83305554"}}, {"type":"entrez-protein","attrs":{"text":"Q9NTJ3","term_id":"30173386"}}, {"type":"entrez-protein","attrs":{"text":"O95347","term_id":"215273886"}}, {"type":"entrez-protein","attrs":{"text":"Q14683","term_id":"29336622"}}, {"type":"entrez-protein","attrs":{"text":"P24928","term_id":"281185484"}}, {"type":"entrez-protein","attrs":{"text":"P30876","term_id":"401012"}}, {"type":"entrez-protein","attrs":{"text":"P51532","term_id":"116242792"}}, {"type":"entrez-protein","attrs":{"text":"O60343","term_id":"67473227"}}, {"type":"entrez-protein","attrs":{"text":"P08195","term_id":"1812588799"}}, {"type":"entrez-protein","attrs":{"text":"P25963","term_id":"126682"}}, {"type":"entrez-protein","attrs":{"text":"Q15653","term_id":"57015399"}}, {"type":"entrez-protein","attrs":{"text":"O00221","term_id":"206729919"}}, {"type":"entrez-protein","attrs":{"text":"Q13501","term_id":"74735628"}}, Strnad P., Zatloukal K., Stumptner C., Kulaksiz H., and Denk H. (2008), Mallory-Denk-bodies: lessons from keratin-containing hepatic inclusion bodies, Zatloukal K., Stumptner C., Fuchsbichler A., Heid H., Schnoelzer M., Kenner L., Kleinert R., Prinz M., Aguzzi A., and Denk H. (2002), p62 Is a common component of cytoplasmic inclusions in protein aggregation diseases, The Mallory body: morphological, clinical and experimental studies (Part 1 of a literature survey), Denk H., Gschnait F., and Wolff K. (1975), Hepatocellar hyalin (Mallory bodies) in long term griseofulvin-treated mice: a new experimental model for the study of hyalin formation, Yokoo H., Harwood T. R., Racker D., and Arak S. (1982), Experimental production of Mallory bodies in mice by diet containing 3,5-diethoxycarbonyl-1,4-dihydrocollidine, Holley A. E., Frater Y., Gibbs A. H., De Matteis F., Lamb J. H., Farmer P. B., and Naylor S. (1991), Isolation of two N-monosubstituted protoporphyrins, bearing either the whole drug or a methyl group on the pyrrole nitrogen atom, from liver of mice given griseofulvin, Ortiz de Montellano P. R., Beilan H. S., and Kunze K. L. (1981), N-Alkylprotoporphyrin IX formation in 3,5-dicarbethoxy-1,4-dihydrocollidine-treated rats. B, Flow-chart of the strategy used to identify potential IB interactors upon ZnPP-induction using biotinylation via antibody recognition (BAR) analyses under denaturing conditions. Reverse transcribed first strand cDNA (1 l) was used in PCRs. carried out the ZnPP in vivo mouse experiments. (42). This product is for research use only, not for diagnostic or therapeutic use. Peak lists were extracted using PAVA, an in-house software developed by UCSF Mass Spectrometry Facility. About ChromoTek - www.chromotek.comChromoTek is a biotech company specialising in molecular tools for bioimaging and proteome analysis. Due to its small size, the GFP-Booster enables higher image quality in epifluorescence, confocal, and super-resolution microscopy: CFP, AcGFP, EGFP, GFP, GFP S65T, mClover, EGFP A206K, pHluorin, PA-GFP, sfGFP, TagGFP, TagGFP2, Citrine, Ecitrine, EYFP, Venus, YFP, Ypet, 10 mM HEPES pH 7.0, 500 mM NaCl, 5 mM EDTA, Preservative: 0.09% sodium azide. The proteins occupying a network hub/node position within their individual clusters, thus could disrupt multiple vital cellular interactions, leading to global hepatic functional collapse. Immunofluorescence analyses of ZnPP-treated cells coupled with 8 M urea/CHAPS-extraction revealed that this IB-loss was due to its sequestration along with IB into insoluble aggregates, thereby releasing NF-B. Bethesda, MD 20894, Web Policies Epitope 302-393 aaApplications: IP/Co-IP, MSFormats: Agarose, Specificity: p53. Generally, pull down experiments have been performed with antibodies. (2006), Conditional disruption of ubiquitous calpains in the mouse, Lazarou M., Sliter D. A., Kane L. A., Sarraf S. A., Wang C., Burman J. L., Sideris D. P., Fogel A. I., and Youle R. J. Both compounds inactivate certain hepatic cytochromes P450, converting their prosthetic heme into N-methylprotoporphyrin(s) (NMPP) that inhibit hepatic ferrochelatase (Fech) resulting in hepatic heme depletion and protoporphyrin IX (PPIX) accumulation (6, 7) (Fig. 5B, supplemental Fig. This email address is being protected from spambots. Considerably higher tissue penetration rates, Superior accessibility and labelling of epitopes in crowded cellular/organelle environments, Less than 2 nm epitope-label displacement minimizes linkage error, Monovalent VHHs do not cluster their epitopes, Validation: structure and function characterized, Consistent and reliable performance due to recombinant production. 5C, ,55D). Fast Your inquiry will be delivered straight to the manufacturer, Secure We only pass your details on to trusted suppliers at your request, Save time Submit your details once and make multiple inquiries, "Best rubustness and ruggudness of equipment". A., and Omary M. B. government site. The supplier does not provide quotations for this product through SelectScience. IB-loss was due to its sequestration into insoluble cytoplasmic aggregates. Bioz Stars score: 86/100, based on 1 PubMed citations. By contrast, upon ZnPP-treatment, despite this normal IB-restoration at 1 h of TNF-treatment, it is apparently functionally defective as a nuclear NF-B-repressor, as NF-B persisted in the nucleus (Fig. We employed high-affinity alpaca Nanobody cross-linked beads (GFP-Trap, ChromoTek, Germany; #gta-20) for IAP, which not only enabled a high-level enrichment of target proteins but also eliminated IgG contaminants that confound downstream LCMS/MS analyses. Latest: Environmental Analysis, Pesticides, Contaminants & Sustainability: Technologies and Latest: 6 upcoming webinars to boost your research, Latest: Materials science: Key technological advancements to celebrate. (2014), A code for RanGDP binding in ankyrin repeats defines a nuclear import pathway, Versatility at the nuclear pore complex: lessons learned from the nucleoporin Nup153, Nakielny S., Shaikh S., Burke B., and Dreyfuss G. (1999), Nup153 is an M9-containing mobile nucleoporin with a novel Ran-binding domain, Partridge J. R., and Schwartz T. U. ASH and NASH patient liver histology reveals that MDBs are associated with inflammatory responses, with MDB-containing hepatocytes often surrounded by neutrophils (1, 9). Proteintech ChromoTek GFP-Booster ATTO647N 17343343 Print Proteintech ChromoTek GFP-Booster ATTO647N GFP VHH conjugated to ATTO 647N. GFP tag, GFP, eGFP, eYFP, CFP, YFP, BFP, GFP-Booster, Green fluorescent protein, AcGFP, GFP S65T, mClover (Clover A260K), Monomeric EGFP A206K, pHluorin (ecliptic), PA-GFP, Superfolder GFP (sfGFP), TagGFP, TagGFP2, Citrine, Ecitrine, Venus, EYFP, Ypet. An official website of the United States government. ChromoTek was founded in 2008 and is located in Martinsried, Germany. B, Venn diagram of proteins enriched in ZnPP-induced aggregates from HEK293T and HepG2 cells indicated 225 proteins as common hits. p62WT and p62KO MEF cells were treated with ZnPP (10 M) or vehicle control for 2 h, and then fixed and stained with anti-IB (green), anti-p62 (magenta) and DAPI (Blue). germany@ptglab.com, Contact Us Nano-Traps are so stable you can literally boil them without compromising function. 1-888-478-4522 proteintech@ptglab.com, (+44) 161 839 3007 Positive GRAVY scores suggested greater hydrophobicity. Cumulative biochemical evidence indicates that in these mouse models as well as in DDC-, NMPP- or PPIX-treated cell models, certain proteins such as p62, cytokeratins CK8/18 and lamin begin to aggregate at a much earlier stage before MDBs are detected (12, 13). Catalog No. The company works closely together with the Center for Integrated Protein Science Munich at the Biocenter of the Ludwig-Maximilian-University Munich. For on-bead digestion after SA pulldown, beads were first resuspended in 6 M urea in 100 mM ammonium bicarbonate (ABC), and reduced by adding 10 mM DTT (final) and incubation at 37 C for 30 min. The .gov means its official. ContactDr. S1C). GFP/RFP-Booster - enhance your fluorescent fusion protein ChromoTek's Booster are very small, highly specific GFP- or RFP-binding proteins covalently coupled to the superior fluorescent dyes from ATTO-TEC. Cells were further washed three times in PBS/0.1% Tween and then mounted using ProLong Diamond Antifade Mountant with DAPI nuclear stain (Molecular Probes, Grand Island, NY). |imprint|terms of use|privacy policy|terms and conditions| Copyright 2013 ChromoTek GmbH. Similarly, many neurodegenerative diseases are also associated with inflammation at an early stage, and amyloid protein aggregation has been shown to initiate an inflammatory response (14). Imprint (Impressum) IB analyses of high salt buffer (HSB)-extracts from ZnPP-treated cell lysates also revealed detectable monomeric and aggregated IB and p62 species, albeit to a much lesser extent than corresponding urea extracts (Fig. Because of inherent commonalities this MDB cell model is a bona fide protoporphyric model, making these findings equally relevant to the liver inflammation associated with clinical protoporphyria. p62 MEF cells: Were generated from p62 KO mice by Prof. Masaaki Komatsu's laboratory (35), Niigata University, Japan, and provided by Prof. Haining Zhu, University of Kentucky. RFP-Trap is used to identify and pull down interaction partners of proteins tagged with RFP (red fluorescent protein) and GFP-Booster restores or increases the GFP (green fluorescent protein) signal in super-resolution microscopy. ChromoTeks lead product, the GFP-Trap, is used for immunoprecipitation (IP, coIP) and/or affinity purification of GFP-tagged proteins. (2001), Heme-regulated eIF2alpha kinase (HRI) is required for translational regulation and survival of erythroid precursors in iron deficiency, Okada K., Yanagawa T., Warabi E., Yamastu K., Uwayama J., Takeda K., Utsunomiya H., Yoshida H., Shoda J., and Ishii T. (2009), The alpha-glucosidase inhibitor acarbose prevents obesity and simple steatosis in sequestosome 1/A170/p62 deficient mice, Komatsu M., Waguri S., Koike M., Sou Y. S., Ueno T., Hara T., Mizushima N., Iwata J., Ezaki J., Murata S., Hamazaki J., Nishito Y., Iemura S., Natsume T., Yanagawa T., Uwayama J., Warabi E., Yoshida H., Ishii T., Kobayashi A., Yamamoto M., Yue Z., Uchiyama Y., Kominami E., and Tanaka K. (2007), Homeostatic levels of p62 control cytoplasmic inclusion body formation in autophagy-deficient mice, Mizushima N., Yamamoto A., Hatano M., Kobayashi Y., Kabeya Y., Suzuki K., Tokuhisa T., Ohsumi Y., and Yoshimori T. (2001), Dissection of autophagosome formation using Apg5-deficient mouse embryonic stem cells, Tan Y., Dourdin N., Wu C., De Veyra T., Elce J. S., and Greer P. A. These score thresholds resulted in a <1% FDR at the peptide level. After ZnPP-treatment, cells were first fixed and then immunostained with IB-antibody, followed by HRP-conjugated secondary antibody. As Nano-Secondaries are recombinantly produced, they are fully sequenced by design, which in combination with high QC standards ensures reliable and stable products virtually without batch-to-batch variation. ChromoTek | LinkedIn ChromoTek's Chromobodies are a novel species of extremely small intracellular functional antibodies. This machinery depends on a direct interaction between the ARPs, RanGDP as well as mobile nucleoporins such as Nup153 and RanBP2 for entry across the NPC into the nucleus.