Journal: Biomacromolecules

Article Title: Intrinsically Fluorescent Oligomeric Cytotoxic Conjugates Toxic for FGFR1-Overproducing Cancers

doi: 10.1021/acs.biomac.1c01280

Figure Lengend Snippet: Impact of engineered GFPp_FGF1 oligomers on FGFR1 binding and activation. (A) Serum-starved NIH3T3 cells were treated with increasing concentrations of the wild-type FGF1 or GFPp_FGF1 oligomers. Cells were lysed, and activation of FGFR1 and receptor-downstream signaling were assessed with Western blotting. The level of tubulin served as a loading control. (B) To determine the kinetics of FGFR1 signaling upon cell stimulation with GFPp_FGF1, serum-starved NIH3T3 cells were stimulated with proteins for up to 6 h. At distinct time points of incubation, cells were lysed and analyzed by Western blotting. (C) Kinetics of the interaction of GFPp_FGF1 oligomers with FGFR1 was analyzed using biolayer interferometry (BLI). The extracellular region of FGFR1 (FGFR1ecd-Fc) was immobilized on Protein A sensors, and then, the receptor was incubated with distinct GFPp_FGF1 oligomers. The association and dissociation profiles were measured.

Article Snippet: Mouse embryo fibroblast cells (NIH3T3) were obtained from American Type Culture Collection (ATCC, Manassas, VA, USA).

Techniques: Binding Assay, Activation Assay, Western Blot, Control, Cell Stimulation, Incubation

Journal: Biomacromolecules

Article Title: Intrinsically Fluorescent Oligomeric Cytotoxic Conjugates Toxic for FGFR1-Overproducing Cancers

doi: 10.1021/acs.biomac.1c01280

Figure Lengend Snippet: Engineering of the fluorescent trimeric cytotoxic conjugate targeting FGFR1. (A) The C-terminal LPETGG sequence was incorporated into the trimeric GFPp_FGF1E via gene synthesis, yielding 3xGFPp_FGF1E_LPETGG. Sortase A recognizes the LPETGG sequence within 3xGFPp_FGF1E_LPETGG and mediates ligation of the tetraglycine peptide-linked MMAE to 3xGFPp_FGF1E_LPETGG, resulting in 3xGFPp_FGF1E_LPET_MMAE. (B) The efficiency of the conjugation and purity of the obtained 3xGFPp_FGF1E_LPET_MMAE were confirmed by SDS-PAGE. (C) The site-specific attachment of MMAE to 3xGFPp_FGF1E_LPETGG was confirmed by MALDI-MS. The impurities that appear in MALDI-MS (about 30,000 Da) are either the result of a minor protein fragmentation during ionization or trace impurities/degradation products not visible in SDS-PAGE and UV spectra but detectable in the high-sensitivity MS approach. (D) Assessment of the biological activity of recombinant proteins. Serum-starved NIH3T3 cells were incubated with FGF1 WT (positive control) or with 3xGFPp_FGF1E_LPETGG and 3xGFPp_FGF1E_LPET_MMAE. Cells were lysed, and activation of FGFR1 was assessed with Western blotting. The level of tubulin served as a loading control. (E) Binding profiles of 3xGFPp_FGF1E_LPETGG and 3xGFPp_FGF1E_LPET_MMAE to FGFR1 were measured using BLI. The extracellular region of FGFR1 (FGFR1ecd-Fc) was immobilized on Protein A sensors and incubated with proteins/conjugates. Association and dissociation profiles were measured.

Article Snippet: Mouse embryo fibroblast cells (NIH3T3) were obtained from American Type Culture Collection (ATCC, Manassas, VA, USA).

Techniques: Sequencing, Ligation, Conjugation Assay, SDS Page, Activity Assay, Recombinant, Incubation, Positive Control, Activation Assay, Western Blot, Control, Binding Assay

Journal: Biomacromolecules

Article Title: Intrinsically Fluorescent Oligomeric Cytotoxic Conjugates Toxic for FGFR1-Overproducing Cancers

doi: 10.1021/acs.biomac.1c01280

Figure Lengend Snippet: Stability analysis of the 3xGFPp_FGF1E_LPETGG and 3xGFPp_FGF1E_LPET_MMAE. (A) 3xGFPp_FGF1E_LPETGG and 3xGFPp_FGF1E_LPET_MMAE were incubated in human serum in the presence of heparin at 37 °C for 96 h. At distinct time points (0, 24, 48, 72, and 96 h), samples were taken, and the oligomeric state of proteins was analyzed using native PAGE UV light imaging. (B) The stability of the GFPp oligomerization scaffold within the trimeric protein and the conjugate was determined by monitoring GFP fluorescence at distinct time points of incubation in human serum at 37 °C. Fluorescence spectra were acquired using a FP-8500 spectrofluorometer (Jasco, Japan) with excitation at 488 nm and emission in the 500–650 nm range. (C) The stability of FGF1E in 3xGFPp_FGF1E_LPETGG and 3xGFPp_FGF1E_LPET_MMAE was determined with Western blotting using antibodies recognizing FGF1. (D) Evaluation of the biological activity of 3xGFPp_FGF1E_LPETGG and its cytotoxic conjugate. Samples were incubated with human serum at 37 °C for 96 h. At distinct time points of incubation (0, 24, 48, 72, and 96 h), proteins were added to serum-starved NIH3T3 cells. Cells were lysed, and activation of FGFR1 and receptor-downstream signaling were assessed with Western blotting. The level of tubulin served as a loading control.

Article Snippet: Mouse embryo fibroblast cells (NIH3T3) were obtained from American Type Culture Collection (ATCC, Manassas, VA, USA).

Techniques: Incubation, Clear Native PAGE, Imaging, Fluorescence, Western Blot, Activity Assay, Activation Assay, Control

Journal:

Article Title: Characterization of the Conformational Alterations, Reduced Anticoagulant Activity, and Enhanced Antiangiogenic Activity of Prelatent Antithrombin *

doi: 10.1074/jbc.M710327200

Figure Lengend Snippet: Isolation of prelatent antithrombin by heparin-agarose chromatography. Left, elution profile of heat-treated native plasma antithrombin after chromatography on a Hi-Trap heparin column (solid line). Protein was eluted from the column with a linear NaCl gradient (dashed line) and was detected by intrinsic fluorescence. The protein peak eluting between 1 and 2 m NaCl was subdivided as shown into two pools, and the leading edge pool was rechromatographed and similarly subdivided to obtain the three pools A, B, and C (A–C), as described under “Experimental Procedures.” Right, elution profiles of antithrombin pools A, B, and C after rechromatography on the Hi-Trap Heparin column using a convex salt gradient (dashed line). Latent antithrombin and untreated native antithrombin were similarly chromatographed. All chromatograms were corrected for background fluorescence by subtracting a chromatogram of buffer alone. Further details are provided under “Experimental Procedures.”

Article Snippet: Reactive loop-cleaved antithrombin was prepared by incubating native antithrombin with human neutrophil elastase (Athens Research Technology) as in previous studies ( 31 , 32 ).

Techniques: Isolation, Chromatography, Clinical Proteomics, Fluorescence

Journal:

Article Title: Characterization of the Conformational Alterations, Reduced Anticoagulant Activity, and Enhanced Antiangiogenic Activity of Prelatent Antithrombin *

doi: 10.1074/jbc.M710327200

Figure Lengend Snippet: Kinetics of conversion of native antithrombin to prelatent and latent forms. A, fluorescence elution profiles obtained after heating native plasma antithrombin at 60 °C in pH 7.4 citrate buffer for the indicated times (in h) and chromatographing 10 μg samples on the Hi-Trap heparin column as in Fig. 1 using the convex salt gradient (dashed line). The different forms of antithrombin eluting from the column were quantitated by integrating the areas under each peak, and the relative amounts of each form were expressed as a percentage of the total fluorescence. Further details are provided under “Experimental Procedures.” B, plot of the time dependence for conversion of native antithrombin (AT) (•) to prelatent (▪), latent (▴), and nonbinding (♦) forms based on the quantitation of these forms in the chromatograms of A and others not shown for clarity. The solid lines indicate the computer fit of data by the model in D along with the fitted rate constants as detailed under “Experimental Procedures.” C, chromatograms of isolated prelatent antithrombin after incubating at 60 °C in pH 7.4 Tris/citrate buffer for the indicated times (in h) and chromatographing on Hi-Trap Heparin with elution using the convex salt gradient (dashed line). Prelatent, latent, and unbound forms of antithrombin were quantitated by integration of peaks and normalizing to the total fluorescence as in A. D, minimal kinetic model consistent with the data of A–C together with the rate constants providing the best fit of the data by this model.

Article Snippet: Reactive loop-cleaved antithrombin was prepared by incubating native antithrombin with human neutrophil elastase (Athens Research Technology) as in previous studies ( 31 , 32 ).

Techniques: Fluorescence, Clinical Proteomics, Quantitation Assay, Isolation

Journal:

Article Title: Characterization of the Conformational Alterations, Reduced Anticoagulant Activity, and Enhanced Antiangiogenic Activity of Prelatent Antithrombin *

doi: 10.1074/jbc.M710327200

Figure Lengend Snippet: Binding of pentasaccharide (H5) and full-length (H26) high affinity heparins to native and prelatent antithrombins Native and prelatent antithrombins were titrated with the indicated high affinity heparins under stoichiometric binding conditions ( I 0.05) or under conditions where K D was well determined ( I 0.15 or I 0.3) as indicated below and in . Binding was monitored from increases in tryptophan fluorescence as in previous studies, and binding parameters were determined by fitting titration curves by the equilibrium binding equation, as described under “Experimental Procedures.” Binding stoichiometries determined in I 0.05 buffer were fixed in fits of titrations in I 0.15 or I 0.3 buffers. Only an upper limit for K D in I 0.05 buffer is provided, because binding was too tight to measure accurately in these titrations. Errors represent ± S.E. for 3–7 titrations.

Article Snippet: Reactive loop-cleaved antithrombin was prepared by incubating native antithrombin with human neutrophil elastase (Athens Research Technology) as in previous studies ( 31 , 32 ).

Techniques: Binding Assay, Fluorescence, Titration

Journal:

Article Title: Characterization of the Conformational Alterations, Reduced Anticoagulant Activity, and Enhanced Antiangiogenic Activity of Prelatent Antithrombin *

doi: 10.1074/jbc.M710327200

Figure Lengend Snippet: Comparison of the kinetics and affinity of the interaction of heparin with native and prelatent antithrombins. A and B compare representative fluorescence titrations of native (•) and prelatent (○) forms of antithrombin (AT) with the heparin pentasaccharide performed under stoichiometric binding conditions (I 0. 05) (A) or under equilibrium binding conditions (I 0.15) (B) as described under “Experimental Procedures.” Titrations were fit by the quadratic binding equation (solid lines) to obtain values for the binding stoichiometry, KD, and the maximal fluorescence change (Table 2). The fitted stoichiometry for the titration at I 0.05 was fixed in fitting the titration at I 0.15. C compares the kinetics of pentasaccharide binding to native (•) and prelatent (○) antithrombins under pseudo-first order conditions as a function of the pentasaccharide concentration. Solid lines are linear regression fits of data from which kon and koff were determined from the slope and intercepts, respectively.

Article Snippet: Reactive loop-cleaved antithrombin was prepared by incubating native antithrombin with human neutrophil elastase (Athens Research Technology) as in previous studies ( 31 , 32 ).

Techniques: Comparison, Fluorescence, Binding Assay, Titration, Concentration Assay

Journal:

Article Title: Characterization of the Conformational Alterations, Reduced Anticoagulant Activity, and Enhanced Antiangiogenic Activity of Prelatent Antithrombin *

doi: 10.1074/jbc.M710327200

Figure Lengend Snippet: SDS and native PAGE characterization of prelatent antithrombin. Shown are the electrophoretic gels of antithrombin pools A, B, and C (5 μg of protein) obtained during the purification of prelatent antithrombin by Hi-Trap heparin chromatography (Fig. 1) under denaturing (SDS) and native conditions. Native and latent antithrombin samples were run as controls. The ability of each antithrombin (AT) pool to form an SDS-stable complex with a molar excess of thrombin (5 μg) is shown in the SDS gel.

Article Snippet: Reactive loop-cleaved antithrombin was prepared by incubating native antithrombin with human neutrophil elastase (Athens Research Technology) as in previous studies ( 31 , 32 ).

Techniques: Clear Native PAGE, Purification, Chromatography, SDS-Gel

Journal:

Article Title: Characterization of the Conformational Alterations, Reduced Anticoagulant Activity, and Enhanced Antiangiogenic Activity of Prelatent Antithrombin *

doi: 10.1074/jbc.M710327200

Figure Lengend Snippet: Stoichiometries and kinetics of inhibition of thrombin and factor Xa by native and prelatent antithrombins Untreated antithrombin (AT) and the different pools obtained from Hi-Trap heparin chromatography of heat-treated antithrombin were compared with respect to (i) the stoichiometries of inhibition (SI) of thrombin (IIa) and (ii) the second order rate constants for the inhibition of thrombin or factor Xa (FXa) in the absence of heparin ( k uncat ) or for the inhibition of factor Xa in the presence of saturating heparin pentasaccharide ( k H5 ) as described under “Experimental Procedures.” Errors represent S.E. obtained from the fits of stoichiometric titrations or reaction kinetic curves as a function of time or heparin concentration.

Article Snippet: Reactive loop-cleaved antithrombin was prepared by incubating native antithrombin with human neutrophil elastase (Athens Research Technology) as in previous studies ( 31 , 32 ).

Techniques: Inhibition, Chromatography, Concentration Assay

Journal:

Article Title: Characterization of the Conformational Alterations, Reduced Anticoagulant Activity, and Enhanced Antiangiogenic Activity of Prelatent Antithrombin *

doi: 10.1074/jbc.M710327200

Figure Lengend Snippet: Antiproliferative activity of prelatent antithrombin. HUVECs were cultured with or without stimulation by FGF-2 and in the absence or presence of prelatent (pool A), latent, and native (untreated and pool C) forms of antithrombin (AT) (see Fig. 1) as indicated for 48 h. The number of viable cells was then assayed colorimetrically. Triplicate assays were performed for each condition, and results were expressed relative to the unstimulated control. Further details are provided under “Experimental Procedures.” Error bars, S.E. values obtained after grouping results from several independent experiments. Prelatent and latent antithrombins produced statistically significant growth inhibition (p < 0.01) at minimal doses of 10 and 50 μg/ml, respectively.

Article Snippet: Reactive loop-cleaved antithrombin was prepared by incubating native antithrombin with human neutrophil elastase (Athens Research Technology) as in previous studies ( 31 , 32 ).

Techniques: Activity Assay, Cell Culture, Control, Produced, Inhibition

Journal:

Article Title: Characterization of the Conformational Alterations, Reduced Anticoagulant Activity, and Enhanced Antiangiogenic Activity of Prelatent Antithrombin *

doi: 10.1074/jbc.M710327200

Figure Lengend Snippet: Comparison of high affinity heparin-catalyzed reactions of native and prelatent antithrombin with proteases. Native (•) or prelatent (○) antithrombins (∼20 nm) were reacted with 1 nm thrombin (left) or 2 nm factor Xa (right) in the presence of variable concentrations of high affinity heparin (HA-heparin) for fixed times of 2 min (thrombin) or 30 s (factor Xa). Thrombin reactions additionally contained 2 mm p-aminobenzamidine to slow the rate sufficiently to allow accurate kinetic measurements. Apparent second order rate constants (kapp) were obtained by dividing observed pseudo-first order rate constants for protease inactivation by the functional antithrombin concentration. Additional corrections were made for the competitive effect of p-aminobenzamidine in reactions with thrombin. The bell-shaped heparin concentration dependence of kapp was fit by the ternary complex model (solid lines for native antithrombin, dashed lines for prelatent antithrombin) to provide values for the binary protein-heparin complex dissociation constants and the true second order rate constants for the reactions of antithrombin-heparin binary complex with each protease (32). These values are tabulated in Table 3. Further details are provided under “Experimental Procedures.”

Article Snippet: Reactive loop-cleaved antithrombin was prepared by incubating native antithrombin with human neutrophil elastase (Athens Research Technology) as in previous studies ( 31 , 32 ).

Techniques: Comparison, Functional Assay, Concentration Assay

Journal:

Article Title: Characterization of the Conformational Alterations, Reduced Anticoagulant Activity, and Enhanced Antiangiogenic Activity of Prelatent Antithrombin *

doi: 10.1074/jbc.M710327200

Figure Lengend Snippet: Kinetics of heparin-catalyzed reactions of native and prelatent antithrombins with thrombin and factor Xa Kinetic parameters were obtained from fits of the data of Figs. ​ Figs.6 6 and for high affinity heparin and low affinity heparin-catalyzed reactions of native and prelatent antithrombins with thrombin or factor Xa by the ternary complex bridging or conformational activation models described under “Experimental Procedures.” The fitted parameters were the rate constant for the reaction of heparin-complexed antithrombin with protease ( k H ) and the dissociation constants for the binary antithrombin-heparin complex ( K AT,H ) and the protease-heparin binary complex ( K Pr,H ).

Article Snippet: Reactive loop-cleaved antithrombin was prepared by incubating native antithrombin with human neutrophil elastase (Athens Research Technology) as in previous studies ( 31 , 32 ).

Techniques: Activation Assay

Journal:

Article Title: Characterization of the Conformational Alterations, Reduced Anticoagulant Activity, and Enhanced Antiangiogenic Activity of Prelatent Antithrombin *

doi: 10.1074/jbc.M710327200

Figure Lengend Snippet: Comparison of low affinity heparin-catalyzed reactions of native and prelatent antithrombins with proteases. Native (•) and prelatent (○) antithrombins were reacted with thrombin (20 nm inhibitor and 1 nm protease) or with factor Xa (50 nm inhibitor and 5 nm protease) in the presence of increasing concentrations of low affinity heparin (LA-heparin) for fixed times of 5 min or for variable reaction times. Apparent second order inactivation rate constants were calculated from observed pseudo-first order rate constants and the functional inhibitor concentration as in Fig. 6. The heparin concentration dependence of kapp was fit by the ternary complex bridging model for reactions with thrombin or by a model in which conformational activation of antithrombin solely contributed to the rate-enhancing effect of heparin for reactions with factor Xa (32). Solid lines indicate the fit of the native antithrombin kinetic data, and dashed lines indicate the fit of the prelatent antithrombin kinetic data.

Article Snippet: Reactive loop-cleaved antithrombin was prepared by incubating native antithrombin with human neutrophil elastase (Athens Research Technology) as in previous studies ( 31 , 32 ).

Techniques: Comparison, Functional Assay, Concentration Assay, Activation Assay

Journal:

Article Title: Characterization of the Conformational Alterations, Reduced Anticoagulant Activity, and Enhanced Antiangiogenic Activity of Prelatent Antithrombin *

doi: 10.1074/jbc.M710327200

Figure Lengend Snippet: Decreased thermal stability of prelatent antithrombin. Shown are melting curves for native (•), prelatent (○), and latent (▵) forms of antithrombin measured from decreases in intrinsic protein fluorescence as a function of increasing temperature (in degrees Kelvin). Data were fit by the van't Hoff equation for a two-state unfolding transition to obtain the melting temperature corresponding to the midpoint of the unfolding curves, as described under “Experimental Procedures.” Fitted melting temperatures are reported in degrees centigrade. The results are representative of three independent experiments.

Article Snippet: Reactive loop-cleaved antithrombin was prepared by incubating native antithrombin with human neutrophil elastase (Athens Research Technology) as in previous studies ( 31 , 32 ).

Techniques: Fluorescence

Journal:

Article Title: Characterization of the Conformational Alterations, Reduced Anticoagulant Activity, and Enhanced Antiangiogenic Activity of Prelatent Antithrombin *

doi: 10.1074/jbc.M710327200

Figure Lengend Snippet: Analysis of conformational alterations in prelatent antithrombin by proteolytic susceptibility. The susceptibility of native, prelatent, and latent forms of antithrombin to digestion by catalytic levels of the nontarget proteases, chymotrypsin and thermolysin (serpin/protease weight ratio of 10:1), was monitored as a function of digestion time by SDS-PAGE as described under “Experimental Procedures.”

Article Snippet: Reactive loop-cleaved antithrombin was prepared by incubating native antithrombin with human neutrophil elastase (Athens Research Technology) as in previous studies ( 31 , 32 ).

Techniques: SDS Page

Journal:

Article Title: Characterization of the Conformational Alterations, Reduced Anticoagulant Activity, and Enhanced Antiangiogenic Activity of Prelatent Antithrombin *

doi: 10.1074/jbc.M710327200

Figure Lengend Snippet: Conformational alterations in prelatent antithrombin probed by 1H NMR spectroscopy. Comparison of 900 MHz 1H NMR spectra of 10 μm samples of native (N), prelatent (P), and latent (L) antithrombins in I 0.15, pH 7.4, D2O buffer at 25 °C. Samples were prepared, and spectra were recorded as described under “Experimental Procedures.” Difference spectra between native and prelatent, native and latent, and prelatent and latent are shown to emphasize the similarity of native and prelatent spectra and the significant differences between either of these forms and latent antithrombin. The spike at 3.7 ppm represents small differences in the H2O content of the samples.

Article Snippet: Reactive loop-cleaved antithrombin was prepared by incubating native antithrombin with human neutrophil elastase (Athens Research Technology) as in previous studies ( 31 , 32 ).

Techniques: Structural Proteomics, Comparison

Journal: PLoS Pathogens

Article Title: Pivotal role for the ESCRT-II complex subunit EAP30/SNF8 in IRF3-dependent innate antiviral defense

doi: 10.1371/journal.ppat.1006713

Figure Lengend Snippet: (A) Immunoblotting of phosphorylated-IRF3 (p-IRF3), SeV, EAP30 and action levels in PH5CH8 cells transfected with control or EAP30 siRNA for 48 h and mock-infected or infected with SeV for additional 8 h. (B) Immunoblotting of IRF3 monomer and dimer forms following native PAGE of the samples shown in (A). (C) Immunoblotting of IRF3, SeV, EAP30, lamin A/C (nuclear protein marker), β-tubulin (cytoplasmic protein marker), and actin loading control in cytoplasmic (CE) and nuclear (NE) fractions of PH5CH8 cells transfected with control or EAP30 siRNA and mock-infected or infected with SeV. (D) Immunoblotting of p-IRF3, actin, ISG56, SeV, and EAP30 in HEK293-shEAP30 and HEK293-shCon cells mock-infected or infected with SeV for 8 h. (E) Whole cells lysate (WCL) were collected from HEK293-shEAP30 and HEK293-shCon cells that were mock-infected or infected with SeV for immunoblotting of CBP, IRF3, EAP30 and actin (left panel) and co-IP analysis of virus-induced CBP-IRF3 association (right panel). (F) ChIP analysis of IRF3 binding to IFNβ, IFNL1, and IFIT1 (ISG56) promoters in nuclear extracts of HEK293-shEAP30 and HEK293-shCon cells that were mock-infected or infected with SeV. The ChIP-enriched DNA levels were analyzed by qPCR and normalized to input DNA, followed by subtraction of nonspecific binding determined using control IgG. “*” denotes statistical differences exist with a P -value of < 0.05.

Article Snippet: PH5CH8 non-neoplastic hepatocytes (provided by Nobuyuki Kato, Okayama University, Japan) [ ], human hepatoma Huh7.5-TLR3 and Huh7-TLR3 cells that were stably reconstituted for the expression of human TLR3 (developed in this laboratory) [ , ], and human embryo kidney HEK293 cells (obtained from American Type Culture Collection), were maintained as described previously [ , , ].

Techniques: Western Blot, Transfection, Control, Infection, Clear Native PAGE, Marker, Co-Immunoprecipitation Assay, Virus, Binding Assay

Journal: PLoS Pathogens

Article Title: Pivotal role for the ESCRT-II complex subunit EAP30/SNF8 in IRF3-dependent innate antiviral defense

doi: 10.1371/journal.ppat.1006713

Figure Lengend Snippet: (A) HEK293 (5x10 4 ) cells were transfected with 100 ng of each plasmid or various double/triple plasmid combinations as indicated, with control vector being added to keep the total amount (300 ng) of transfected DNA constant in each condition. 48 h later, cells were challenged with VSV-Luc (MOI = 0.1) for 6 h (except the mock group) followed by cell lysis and luciferase assay. (B) Immunoblot analysis of transfected EAP30, EAP20 (using anti-HA), CBP, IRF3 (using anti-FLAG) and endogenous actin under experimental conditions of (A) for each transfection groups. (C-E) qPCR analysis of IFN-β (C), OASL (D), and IFN-λ1 (E) mRNA levels in HEK293 cells transfected with different plasmid combinations similar to panel (A) for 48 h. “*”, “**”, and “***” denote statistical differences exist as compared with empty vector-transfected cells with a P -value of < 0.05, < 0.01, and <0.001, respectively.

Article Snippet: PH5CH8 non-neoplastic hepatocytes (provided by Nobuyuki Kato, Okayama University, Japan) [ ], human hepatoma Huh7.5-TLR3 and Huh7-TLR3 cells that were stably reconstituted for the expression of human TLR3 (developed in this laboratory) [ , ], and human embryo kidney HEK293 cells (obtained from American Type Culture Collection), were maintained as described previously [ , , ].

Techniques: Transfection, Plasmid Preparation, Control, Lysis, Luciferase, Western Blot

Journal: eLife

Article Title: Secreted dengue virus NS1 from infection is predominantly dimeric and in complex with high-density lipoprotein

doi: 10.7554/eLife.90762

Figure Lengend Snippet: DENV 2 WT cell culture supernatant was filtered, supplemented with protease inhibitor cocktail and 0.05% sodium azide, concentrated using a 100 kDa MWCO Vivaflow cassette and purified using 56.2 anti-NS1 antibody immunoaffinity chromatography. The eluted isNS1wt was dialyzed against PBS, concentrated, and stored at –80°C until further use. ( a ) Schematic of isNS1 purification to illustrate the samples used for gel analyses. % NS1 is measured by the total amount of NS1 (quantified using the anti-NS1 ELISA kit [Bio-Rad] as a percentage of total protein [quantified using the Bradford assay]) found in each sample. Details of the % enrichment in NS1 along the purification process are as shown in . ( b ) Coomassie blue detection of proteins from crude, wash, and elute immunoaffinity fractions for isNS1wt, with the recombinant sNS1 (rsNS1) obtained from as a positive control, after separation on a 10% Native-PAGE gel (left). The crude and elute fractions contain 1 µg of total protein. The wash fraction contains approximately 100 ng of total protein in maximum well volume of the gel. The same set of samples were also subjected to a western blot detection of NS1 using 56.2 anti-NS1 antibody after separation on a 10% Native-PAGE (right). The crude and elute fractions contain 500 ng of total protein. The wash fraction contains approximately 100 ng of total protein in maximum well volume of the gel. ( c ) Coomassie blue detection of proteins from crude, wash, and elute immunoaffinity fractions for isNS1wt and rsNS1 , after separation on a 4–20% reducing SDS-PAGE gel. The crude and elute fractions contain 1 µg of total protein. The wash fraction contains approximately 100 ng of total protein in maximum well volume of the gel. Similarly, the same set of samples were also subjected to a western blot detection of NS1 and ApoA1 using 56.2 anti-NS1 antibody or ApoA1 antibody (Biorbyt, orb10643), respectively, after separation on a 4–20% reducing SDS-PAGE (right). ( d ) In-gel protein identification of the purified isNS1wt by liquid chromatography mass spectrometry (LC-MS). Proportion of NS1, ApoA1 and other unidentified proteins quantified in total ion intensity, obtained from the following samples: elute in solution (boxed in blue), 250 kDa gel band (boxed in purple), 50 kDa gel band (boxed in red), and 25 kDa gel band (green). The boxed gel bands are from representative gels showing the different protein species found while the actual gel bands used for protein identification by LC-MS are as shown in . Figure 1—source data 1. Raw and annotated image for the PAGE gel stained in Coomassie blue. Figure 1—source data 2. Raw and annotated image for the western blot analysis (anti-NS1). Figure 1—source data 3. Raw and annotated image for the PAGE gel stained in Coomassie blue in . Figure 1—source data 4. Raw and annotated image for the western blot analysis (anti-NS1) in . Figure 1—source data 5. Raw and annotated image for the western blot analysis (anti-ApoA1) in .

Article Snippet: 75 μL of AG agarose beads was pre-coupled with either 10 μg or 50 μg of rabbit polyclonal ApoA1 Ab (Biorbyt, orb10643).

Techniques: Cell Culture, Protease Inhibitor, Purification, Chromatography, Enzyme-linked Immunosorbent Assay, Bradford Assay, Recombinant, Positive Control, Clear Native PAGE, Western Blot, SDS Page, Liquid Chromatography, Mass Spectrometry, Liquid Chromatography with Mass Spectroscopy, Staining

Journal: eLife

Article Title: Secreted dengue virus NS1 from infection is predominantly dimeric and in complex with high-density lipoprotein

doi: 10.7554/eLife.90762

Figure Lengend Snippet: ( a ) Schematic of the isNS1 batch immunoaffinity purification protocol and the downstream analyses used in this article. Infected cell supernatant from Vero cells (either WT or T164S EDEN2) was harvested at 72 hpi, clarified, filtered, supplemented with a protease inhibitor cocktail and 0.05% sodium azide, and finally concentrated using a 100 kDa MWCO Vivaflow cassette attached to a peristaltic pump. isNS1wt or isNS1ts was then batch immunoaffinity purified using the 56.2 anti-NS1 antibody immobilized on the AminoLink resin. The resin was then loaded into a column, washed with at least 10 CV of PBS (pH 7.4), eluted with 0.1 M glycine (pH 2.7), and immediately neutralized with 1 M Tris-HCl (pH 9.0). The eluted protein was then dialyzed against PBS and concentrated using a 100 kDa MWCO Amicon ultracentrifugal unit and stored at –80°C before use. The protein purity was determined via Coomassie blue after separation on a reducing SDS-PAGE. The protein bands observed on the gel were then validated in a western blot against NS1 and ApoA1. Protein quality was also determined via NS1 western blot following separation on a Native-PAGE. Excised bands corresponding to 250 kDa on the Native gel, and 50 kDa and 25 kDa bands on the denatured gel as well as the elute in solution were also subjected to protein identification via liquid chromatography mass spectrometry (LC-MS). Purified isNS1wt and isNS1ts were separately complexed with Fab56.2 and Ab56.2 and analyzed through an analytical size exclusion chromatography to ensure formation of stable complexes for imaging via electron microscopy. Purified isNS1wt and isNS1ts were also crosslinked with disuccinimidyl sulfoxide (DSSO) to determine interaction sites between isNS1 and ApoA1 via LC-MS. ( b ) Enrichment of NS1 during the immunoaffinity purification process. isNS1wt purification is used as a representative for the isNS1 purification process. The proportion of NS1 over total protein was measured by taking a percentage of the total amount of NS1 measured (using NS1 ELISA) out of the total protein measured (using Bradford assay) in the crude supernatant before concentrating, crude supernatant after concentrating, immunoaffinity PBS wash and finally in the immunoaffinity elute after buffer exchange against PBS and further concentrating. There is an approximately 455-fold enrichment of NS1 from the concentrated crude supernatant to the immunoaffinity elute. ( c ) Representative negative stain electron micrograph of isNS1 image on a 120 kV FEI Tecnai T12 equipped with an Eagle 4 mega pixel CCD camera. The corresponding 2D classes from the particles picked are shown on the right, red arrows highlighting the NS1 dimer protruding out of the spherical density.

Article Snippet: 75 μL of AG agarose beads was pre-coupled with either 10 μg or 50 μg of rabbit polyclonal ApoA1 Ab (Biorbyt, orb10643).

Techniques: Immunoaffinity Purification, Infection, Protease Inhibitor, Purification, SDS Page, Western Blot, Clear Native PAGE, Liquid Chromatography, Mass Spectrometry, Liquid Chromatography with Mass Spectroscopy, Size-exclusion Chromatography, Imaging, Electron Microscopy, Enzyme-linked Immunosorbent Assay, Bradford Assay, Buffer Exchange, Staining

Journal: eLife

Article Title: Secreted dengue virus NS1 from infection is predominantly dimeric and in complex with high-density lipoprotein

doi: 10.7554/eLife.90762

Figure Lengend Snippet: ( a ) Coomassie blue detection of proteins of immunoaffinity-purified isNS1wt and isNS1ts (elute) after separation on a 10% Native-PAGE. The 250 kDa bands (purple) that were present in both isNS1wt and isNS1ts, as well as the 100 kDa band (pink) that was present only in isNS1ts, were excised for protein identification analyses via LC-MS. ( b ) Coomassie blue detection of proteins of immunoaffinity-purified isNS1wt and isNS1ts (elute) after separation on a 10% reducing SDS-PAGE. The 50 kDa bands (red) and the 25 kDa bands (green) in both isNS1wt and isNS1ts elutes were excised for protein identification analyses via LC-MS. ( c ) Label-free quantification (LFQ) of NS1, ApoA1 and other unidentified proteins in total ion intensity via LC-MS (n = 3) in the following samples from isNS1wt (top table) and isNS1ts (bottom table): elute in solution, 250 kDa gel band (boxed in purple), 100 kDa gel band (boxed in pink; present only in isNS1ts), 50 kDa gel band (boxed in red), and 25 kDa gel band (green). Figure 1—figure supplement 2—source data 1. Raw and annotated image for the PAGE gel stained in Coomassie blue for isNS1wt. Figure 1—figure supplement 2—source data 2. Raw and annotated image for the PAGE gel stained in Coomassie blue for isNS1ts. Figure 1—figure supplement 2—source data 3. Raw and annotated image for the SDS-PAGE gel stained in Coomassie blue for isNS1wt and isNS1ts presented in .

Article Snippet: 75 μL of AG agarose beads was pre-coupled with either 10 μg or 50 μg of rabbit polyclonal ApoA1 Ab (Biorbyt, orb10643).

Techniques: Purification, Clear Native PAGE, Liquid Chromatography with Mass Spectroscopy, SDS Page, Quantitative Proteomics, Staining

Journal: eLife

Article Title: Secreted dengue virus NS1 from infection is predominantly dimeric and in complex with high-density lipoprotein

doi: 10.7554/eLife.90762

Figure Lengend Snippet: ( a ) Coomassie blue detection of proteins from 1 µg of total protein of crude and elute, and 100 ng of total protein (in maximum well volume) in wash immunoaffinity fractions (low concentration due to large volume), with the recombinant sNS1 (rsNS1) obtained from as a positive control, after separation on a 10% Native-PAGE gel (left). The same set of samples were also subjected to a western blot detection of NS1 using Ab56.2, in 500 ng of total protein (except for wash where 100 ng in the maximum well volume) after separation on a 10% Native-PAGE (right). ( b ) Coomassie blue detection of proteins from 1 μg of total protein of crude and elute, and 100 ng of total protein (maximum well volume) in wash immunoaffinity fractions for isNS1ts and rsNS1, after separation on a 4–20% reducing SDS-PAGE gel. 1 µg of total protein in the same set of samples (except for wash where 100 ng in the maximum well volume) were also subjected to a western blot detection of NS1 and ApoA1 using Ab56.2 or ApoA1 antibody (Biorbyt, orb10643), respectively, after separation on a 4–20% reducing SDS-PAGE (right). ( c ) In-gel protein identification of the purified isNS1ts by liquid chromatography mass spectrometry (LC-MS). Proportion of NS1, ApoA1, and other unidentified proteins quantified in total ion intensity, obtained from the following samples: elute in solution (boxed in blue), 250 kDa gel band (boxed in purple), 50 kDa gel band (boxed in red), and 25 kDa gel band (green). The boxed gel bands are from representative gels showing the different protein species found while the actual gel bands used for protein identification by LC-MS are as shown in .

Article Snippet: 75 μL of AG agarose beads was pre-coupled with either 10 μg or 50 μg of rabbit polyclonal ApoA1 Ab (Biorbyt, orb10643).

Techniques: Concentration Assay, Recombinant, Positive Control, Clear Native PAGE, Western Blot, SDS Page, Purification, Liquid Chromatography, Mass Spectrometry, Liquid Chromatography with Mass Spectroscopy

Journal: eLife

Article Title: Secreted dengue virus NS1 from infection is predominantly dimeric and in complex with high-density lipoprotein

doi: 10.7554/eLife.90762

Figure Lengend Snippet: ( a ) SDS-PAGE analysis of isNS1wt with or without the addition of disuccinimidyl sulfoxide (DSSO) crosslinker. ( b ) The identified crosslinks are visualized on the NS1 and bovine ApoA1 constructs. The intramolecular (NS1:NS1 and ApoA1:ApoA1) crosslinks are in magenta. The intermolecular NS1:ApoA1 crosslinks are in green. ( c ) The isNS1 residues that are involved in NS1:NS1 interactions are visualized on the NS1 dimer model. The NS1 cartoon model is colored by its three domains, namely the β-roll (orange), wing (blue), and β-ladder (cyan) with the intramolecular (magenta) and intermolecular (green) crosslinking sites depicted as spheres. ( d ) The overall model interpretation of NS1:ApoA1 complex within the crosslinker theoretical distance cutoff at <30 Å as depicted. ApoA1 dimer cartoon model with its conserved helices as labeled colored in intervals of gray and light purple. ( e ) The NS1:ApoA1 dimer model with validated crosslinks was fitted into the cryoEM envelope. ( f ) SDS-PAGE analysis of crosslinked rsNS1 alone or with human high-density lipoprotein (HDL) (lanes 3–5). Non-crosslinked rsNS1 and human HDL are the control (lanes 1–2). The crosslinked rsNS1 can be seen in higher oligomers (lane 3). ( g ) Identified crosslinks are mapped on the NS1 and ApoA1 constructs, colored as per panel ( b ). Figure 4—source data 1. Raw and annotated image for the PAGE gel visualized using silver stain. Figure 4—source data 2. Raw and annotated image for the PAGE gel visualized using silver stain.

Article Snippet: 75 μL of AG agarose beads was pre-coupled with either 10 μg or 50 μg of rabbit polyclonal ApoA1 Ab (Biorbyt, orb10643).

Techniques: SDS Page, Construct, Labeling, Control, Silver Staining

Journal: eLife

Article Title: Secreted dengue virus NS1 from infection is predominantly dimeric and in complex with high-density lipoprotein

doi: 10.7554/eLife.90762

Figure Lengend Snippet: 2.4 µM of total protein in the purified isNS1wt1complex and 5.8 µM of total protein in the purified isNS1ts complex were crosslinked with disuccinimidyl sulfoxide (DSSO) in an isNS1: DSSO molar ratio of 1:100 for 45 min at room temperature. The crosslinking reaction was then quenched with 20 mM Tris-HCl (pH 8.0) for 15 min at room temperature. Uncrosslinked and crosslinked isNS1wt and isNS1ts were ran under reducing (with DTT) and non-reducing (without DTT) conditions in a SDS-PAGE ( a ) as well as under Native conditions in a Native-PAGE ( b ). The gels were subjected to both silver stain and an NS1 western blot to validate NS1 bands seen in the silver-stained gels. ( c ) The residue map of sNS1ts and ApoA1 showing the intra- (magenta) and inter- (green) molecular crosslinks. The sNS1 β-roll, wing, and β-ladder domains are colored in orange, blue, and cyan. The repeated alpha helices of ApoA1 are colored in alternating gray and light purple. ( d ) Inter- and intramolecular crosslinks identified for NS1ts:ApoA1 complex. The residues involved in the intramolecular crosslinks are depicted as magenta spheres and those in intermolecular crosslinks are in red. ( e ) Rigid-body fitting of the overall model into the Fab-isNS1ts-HDL density map in gray and contoured at 0.17. ( f ) Superimposition between isNS1wt and isNS1ts intra- and intermolecular crosslinks. isNS1ts inter- and intramolecular crosslinks are depicted as magenta and red spheres while the isNS1wt intra- and intermolecular crosslinks are in cyan and blue spheres, respectively. Figure 4—figure supplement 1—source data 1. Raw and annotated image for the western blot analysis (anti-NS1) on an SDS-PAGE gel. Figure 4—figure supplement 1—source data 2. Raw and annotated image for the western blot analysis (anti-NS1) on a Native gel.

Article Snippet: 75 μL of AG agarose beads was pre-coupled with either 10 μg or 50 μg of rabbit polyclonal ApoA1 Ab (Biorbyt, orb10643).

Techniques: Purification, SDS Page, Clear Native PAGE, Silver Staining, Western Blot, Staining, Residue

Journal: eLife

Article Title: Secreted dengue virus NS1 from infection is predominantly dimeric and in complex with high-density lipoprotein

doi: 10.7554/eLife.90762

Figure Lengend Snippet: ( a ) Cartoon representation of isNS1 hexamer due to crystal packing (PDB:4O6B). ( b ) Cartoon representation of isNS1 hexamer cryoEM model with white transparent map and black outline (EMDB: 32843). The intra- and intermolecular crosslinked residues are in magenta and green spheres. The light orange sphere with 30 Å radius represents the maximum range of crosslink-able Cα atoms from Ser2 from β-roll. ( c ) Cartoon representation of sNS1 dimer–ApoA1 cryoEM model from this study. For all panels, the top view (left panel) and side view (right panel) with intra- and intermolecular crosslinks residues in magenta and green spheres respectively on one dimer of sNS1. Domains from the sNS1 dimer molecule are colored in orange (β-roll), blue (wing), and cyan (β-ladder). The light orange sphere has a radius of 30 Å and represent the maximum range between the Cα atoms that can be crosslinked to Ser2 residue from β-roll.

Article Snippet: 75 μL of AG agarose beads was pre-coupled with either 10 μg or 50 μg of rabbit polyclonal ApoA1 Ab (Biorbyt, orb10643).

Techniques: Residue

Journal: eLife

Article Title: Secreted dengue virus NS1 from infection is predominantly dimeric and in complex with high-density lipoprotein

doi: 10.7554/eLife.90762

Figure Lengend Snippet: ( a ) AG129 mice (n = 10) were infected with DENV2 NS1 T164S mutant virus and the pooled infected sera collected on day 4 post-infection was subjected to sNS1 immunoaffinity purification using anti-NS1 56.2 coupled resin as in . 2 mg of the purified eluate was then subjected to western blot analysis after separation and transfer from Native-PAGE for detection of ApoA1 and NS1. ApoA1 was detected using the mouse monoclonal anti-ApoA1 clone 513 (Invitrogen, MIA1404) (left panel) and the oligomeric NS1 was detected using anti-NS1 56.2 IgG clone (right panel). ( b ) Protein AG resin (Pierce) pre-cleared DENV1-infected patient serum (n = 1) from the CELADEN trial was immunoprecipitated with 10 or 50 mg of rabbit polyclonal anti-ApoA1 antibody (Biorbyt, orb10643) to detect association between ApoA1 and NS1 by ELISA. The amount of ApoA1 and NS1 in the immunoprecipitated sample was determined by human ApoA1 (Abcam, ab189576) and Platelia NS1 Ag (Bio-Rad) ELISAs. Figure 7—source data 1. Raw and annotated image for the western blot analysis (anti-NS1 and anti-apoA1) on a Native gel.

Article Snippet: 75 μL of AG agarose beads was pre-coupled with either 10 μg or 50 μg of rabbit polyclonal ApoA1 Ab (Biorbyt, orb10643).

Techniques: Infection, Mutagenesis, Virus, Immunoaffinity Purification, Purification, Western Blot, Clear Native PAGE, Immunoprecipitation, Enzyme-linked Immunosorbent Assay

Journal: eLife

Article Title: Secreted dengue virus NS1 from infection is predominantly dimeric and in complex with high-density lipoprotein

doi: 10.7554/eLife.90762

Figure Lengend Snippet: The different helices of ApoA1 are as annotated above the aligned sequences. The residues highlighted in cyan are the crosslinked residues. The asterisk (*) signifies positions that have a fully conserved residue, the colon (:) signifies conservation between groups of strong similar properties (scoring >0.5 in the Gonnet PAM 250 matrix) and the period (.) signifies conservation between groups of weakly similar properties (scoring <0.5 in the Gonnet PAM 250 matrix) while a blank signifies dissimilar residues. Crosslinked residues identified from crosslinking mass spectrometry (XL-MS) are highlighted in cyan. There is a sequence similarity of 78.65% between bovine and human ApoA1, 65.17% between mouse and human ApoA1, and 66.79% between bovine and mouse ApoA1 (calculated by multiple sequence alignment, NCBI protein blast). Generated with Clustal Omega v1.2.4.

Article Snippet: 75 μL of AG agarose beads was pre-coupled with either 10 μg or 50 μg of rabbit polyclonal ApoA1 Ab (Biorbyt, orb10643).

Techniques: Residue, Mass Spectrometry, Structural Proteomics, Sequencing, Generated