Journal: The Journal of Immunology Author Choice

Article Title: Circulating Plasma Extracellular Vesicles from Septic Mice Induce Inflammation via MicroRNA- and TLR7-Dependent Mechanisms

doi: 10.4049/jimmunol.1801008

Figure Lengend Snippet: Plasma EVs from septic mice induce cytokine production through TLR7-MyD88 signaling. BMDM were isolated from WT and genetically modified mice (TLR7−/−, TLR3−/−, MyD88−/−, Trif−/−) and treated with sham EVs (20 μg/ml), CLP EVs (20 μg/ml), P3C (TLR2 ligand, 1 μg/ml), poly(I:C) (TLR3 ligands, 10 μg/ml), or R837 (TLR7 ligand, 0.25 μg/ml). Sixteen hours after the treatment, the culture media were collected and cytokines (MIP-2, IL-6) were measured by ELISA. (A and B) TLR7 but not TLR3 signaling contributed to CLP EV–induced cytokine production. (C and D) MyD88- but not Trif-deficiency abolished CLP EV–mediated MIP-2 and IL-6 production. Each bar represents triplicate samples with each experiment repeated twice. ***p < 0.001, #p < 0.0001, versus PBS group.

Article Snippet: Bone marrow–derived Mϕs (BMDM) were incubated in serum-free culture medium containing 0.05% BSA for 1 h before treatment with EVs (20 μg/ml), poly(I:C) (10 μg/ml; Enzo Life Sciences, Farmingdale, NY), R837 (1μg/ml; InvivoGen, San Diego, CA), or Pam3Cys (P3C; 1μg/ml; Enzo Life Sciences) for overnight.

Techniques: Isolation, Genetically Modified, Enzyme-linked Immunosorbent Assay

Journal: Frontiers in Immunology

Article Title: Targeting Toll-Like Receptors: Promising Therapeutic Strategies for the Management of Sepsis-Associated Pathology and Infectious Diseases

doi: 10.3389/fimmu.2013.00387

Figure Lengend Snippet: Selection of clinical trials testing drugs targeting TLRs for bacterial and viral infections .

Article Snippet: FluMist + Poly(I:C 12 U) , Hemispherx Biopharma , TLR3 , Agonist , Randomized, double-blind phase 1 and phase 2 , NCT01591473 , Immunogenicity and safety of human influenza vaccine in healthy volunteers , Currently recruiting.

Techniques: Selection, Clinical Proteomics, Immunopeptidomics

Journal: Nature Communications

Article Title: USP52 acts as a deubiquitinase and promotes histone chaperone ASF1A stabilization

doi: 10.1038/s41467-018-03588-z

Figure Lengend Snippet: Histone chaperone ASF1A is physically associated with USP52. a Immunoaffinity purification and mass spectrometry analysis of ASF1A-containing protein complexes. MCF-7 cells allow doxycycline (Dox)-inducible expression of stably integrated FLAG-ASF1A was generated. Whole-cell extracts from MCF-7 cells with or without FLAG-ASF1A expression were prepared and subjected to affinity purification using an anti-FLAG affinity column. After extensive washing, the bound proteins were eluted with excess FLAG peptides, resolved, and then visualized by silver staining on SDS-PAGE. The protein bands on the gel were recovered by trypsinization and analyzed by mass spectrometry. Detailed results from the mass spectrometric analysis are provided as Supplementary Data . b Whole-cell lysates from HeLa or MCF-7 cells were immunoprecipitated (IP) followed by immunoblotting (IB) with antibodies against the indicated proteins. c ASF1A-containing protein complex purified from HeLa cells stably expressing FLAG-ASF1A was fractionated by fast protein liquid chromatography (FPLC) on Superose 6 size exclusion columns with high salt buffer. Chromatographic elution profiles and IB analysis of the chromatographic fractions with antibodies against the indicated proteins are shown. The elution positions of calibration proteins with known molecular masses are indicated, and an equal volume from each fraction was analyzed. d Co-immunoprecipitation analysis of the interaction between USP52 and ASF1A with cellular lysates from different cellular compartments of MCF-7 cells. e Immunostaining and confocal microscopy analysis of USP52 and ASF1A subcellular localization in MCF-7 cells. Scale bar, 10 μm. f Co-immunoprecipitation analysis of the molecular interface between ASF1A and USP52 with cellular lysates from HeLa cells expressing FLAG-GFP tagged full length or deletions of USP52. The conserved domain of USP52 was determined by the SMART program. WD40: WD40 repeat domain, UCH: ubiquitin C-terminal hydrolase domain, EXO: exonuclease domain. g Co-immunoprecipitation analysis of the molecular interface between ASF1A and USP52 with cellular lysates from HeLa cells expressing FLAG-tagged full length or deletions of ASF1A

Article Snippet: K6-linked tetra-ubiquitin chains (UC-15), K11-linked tetra-ubiquitin chains (UC-45), K29-linked tetra-ubiquitin chains (UC-83), K33-linked tetra-ubiquitin chains (UC-103), K48-linked tetra-ubiquitin chains (UC-210B), K63-linked tetra-ubiquitin chains (UC-310B), M1-linked tetra-ubiquitin chains (UC-710B), K2-linked di-ubiquitin chains (UC-61B) and K48-linked di-ubiquitin chains (UC-200B) were purchased from Boston Biochem.

Techniques: Immunoaffinity Purification, Mass Spectrometry, Expressing, Stable Transfection, Generated, Affinity Purification, Affinity Column, Silver Staining, SDS Page, Immunoprecipitation, Western Blot, Purification, Fast Protein Liquid Chromatography, Immunostaining, Confocal Microscopy

Journal: Nature Communications

Article Title: USP52 acts as a deubiquitinase and promotes histone chaperone ASF1A stabilization

doi: 10.1038/s41467-018-03588-z

Figure Lengend Snippet: USP52 is a deubiquitinase. a In vitro deubiquitination assays with E . coli cells-purified UCH domain of USP52 (USP52/UCH, 1 μg) and K48-linked tetra-ubiquitin linkages (1 μg) under different time points as indicated. The cleavage effect was examined by western blotting with antibody against ubiquitin. The asterisk indicates the recombinant protein stained by Commassie Blue. b In vitro deubiquitination assays with Sf9 cells-purified USP52/UCH (1 μg) and K48-linked tetra-ubiquitin linkages (1 μg) under different time points as indicated. The cleavage effect was examined by western blotting with antibody against ubiquitin. The asterisk indicates the recombinant protein stained by Commassie Blue. c In vitro deubiquitination assays with different types of ubiquitin linkages and increasing amounts of Sf9 cells-purified USP52/UCH (1 and 3 μg) or control eluents. After 4 h of incubation, the cleavage effect was examined by western blotting with antibody against ubiquitin. d In vitro deubiquitination assays with different types of ubiquitin linkages (1 μg) and HeLa cells-purified full-length USP52 (1 μg) or UCH domain deficient USP52 (USP52/∆UCH, 1 μg) with high salt and detergent buffer. After 4 h of incubation, the cleavage effect was examined by western blotting with antibody against ubiquitin. The asterisk indicates the recombinant protein stained by silver staining. e In vitro deubiquitination assays with K48- or K63-linked tetra-ubiquitins (0.5 μg) and Sf9 cells-purified USP52/UCH (3 μg) in the presence or absence of 2 mM alkylating reagent N -ethylmaleimide (NEM). Sf9 cells-purified recombinant C-terminal USP7 (UCH domain and C-terminal ubiquitin-like domain, 1.5 μg) was taken as a positive control. After 4 h of incubation, the cleavage effect was examined by western blotting with antibody against ubiquitin. f In vitro deubiquitination assays with K48- or K63-linked tetra-ubiquitins (0.5 μg) and Sf9 cells-purified USP52/UCH (3 μg) or cysteine mutant of UCH domain (C528A/C530A, 3 μg). After 4 h of incubation, the cleavage effect was examined by western blotting with antibody against ubiquitin. CA represents mutant carrying both C528A and C530A (C528A/C530A)

Article Snippet: K6-linked tetra-ubiquitin chains (UC-15), K11-linked tetra-ubiquitin chains (UC-45), K29-linked tetra-ubiquitin chains (UC-83), K33-linked tetra-ubiquitin chains (UC-103), K48-linked tetra-ubiquitin chains (UC-210B), K63-linked tetra-ubiquitin chains (UC-310B), M1-linked tetra-ubiquitin chains (UC-710B), K2-linked di-ubiquitin chains (UC-61B) and K48-linked di-ubiquitin chains (UC-200B) were purchased from Boston Biochem.

Techniques: In Vitro, Purification, Western Blot, Recombinant, Staining, Incubation, Silver Staining, Positive Control, Mutagenesis

Journal: Nature Communications

Article Title: USP52 acts as a deubiquitinase and promotes histone chaperone ASF1A stabilization

doi: 10.1038/s41467-018-03588-z

Figure Lengend Snippet: USP52 opposes ASF1A polyubiquitination. a In vitro deubiquitination assays with HeLa cells-purified Myc-USP52 (1, 2, and 3 μg for each lane) and HA-Ub-conjugated FLAG-ASF1A with high salt and detergent buffer. b HeLa cells stably expressing FLAG-ASF1A were co-transfected with control siRNA or USP52 siRNA together with HA-Ub as indicated. Cellular extracts were prepared for co-immunoprecipitation assays with anti-FLAG followed by IB with anti-HA. c HeLa cells stably expressing FLAG-ASF1A were co-transfected with HA-Ub and different amounts of Myc-USP52. Cellular extracts were prepared for co-immunoprecipitation assays with anti-FLAG followed by IB with anti-HA. d HeLa cells were transfected with different amounts of Myc-USP52 and cellular extracts were prepared for co-immunoprecipitation assays with anti-ASF1A followed by IB with antibody against ubiquitin. e Cellular extracts from HeLa cells expressing HA-Ub/K48-only and different amounts of Myc-USP52 were prepared for co-immunoprecipitation assays with anti-ASF1A followed by IB with anti-HA. f In vitro deubiquitination assays with HeLa cells-purified FLAG-tagged ASF1A-Ub/K48-only and different amounts of Myc-USP52 (1, 2, and 3 μg for each lane) with high salt and detergent buffer. g HeLa cells stably expressing wild-type ASF1A (ASF1A/wt) or different K to R mutants were co-transfected with HA-Ub and control vector or Myc-USP52 as indicated. Cellular extracts were prepared for co-immunoprecipitation assays with anti-FLAG followed by IB with anti-HA. h Mass spectrometry analysis of ASF1A ubiquitin conjugation sites. HeLa cells stably expressing FLAG-ASF1A were co-transfected with HA-Ub and USP52 siRNA. Cellular extracts were collected and sequentially purified with anti-FLAG affinity gel and HA affinity gel to enrich HA-Ub-conjugated ASF1A. After trypsinization, the retrieved peptides were subjected to mass spectrometry analysis. Fragmentation spectrums and parameters of the identified ASF1A peptides with di-Glycine remnant are shown. Detailed results from the mass spectrometric analysis are provided as Supplementary Data

Article Snippet: K6-linked tetra-ubiquitin chains (UC-15), K11-linked tetra-ubiquitin chains (UC-45), K29-linked tetra-ubiquitin chains (UC-83), K33-linked tetra-ubiquitin chains (UC-103), K48-linked tetra-ubiquitin chains (UC-210B), K63-linked tetra-ubiquitin chains (UC-310B), M1-linked tetra-ubiquitin chains (UC-710B), K2-linked di-ubiquitin chains (UC-61B) and K48-linked di-ubiquitin chains (UC-200B) were purchased from Boston Biochem.

Techniques: In Vitro, Purification, Stable Transfection, Expressing, Transfection, Immunoprecipitation, Plasmid Preparation, Mass Spectrometry, Conjugation Assay