Journal: PLoS ONE

Article Title: A Putative Non-Canonical Ras-Like GTPase from P . falciparum : Chemical Properties and Characterization of the Protein

doi: 10.1371/journal.pone.0140994

Figure Lengend Snippet: The depicted plot shows the absolute fluorescense.of the formed PfG-Protein-BGTP complex monitored at a wavelength between 480 nm and 510 nm versus increasing protein concentrations with a crude extract of the purified expressed PfG- protein (black square) and a purified enzyme preparation (black circle) after native purificaion. A non-recombinant pET-28a vector (open triangle) and a constitutively expressed Gα s human subunit (open square) were employed as a positive and a negative control. Each point represents the mean value of three different experiments.

Article Snippet: Fluorescense exitation (470 nm) and emission (510 nm) were determined in a fluorescense microplate reader (GoMax-Multi, Promega, Karlsruhe).

Techniques: Purification, Recombinant, Plasmid Preparation, Negative Control

Journal: PLoS ONE

Article Title: A Putative Non-Canonical Ras-Like GTPase from P . falciparum : Chemical Properties and Characterization of the Protein

doi: 10.1371/journal.pone.0140994

Figure Lengend Snippet: Part A Absolute Fluorescense of binding of 50 nmol BODIPY FL GTPγS to the plasmodial, PfG-protein (black line). The increase in fluorescence was monitored until saturation over a time interval of 600 s. Part B At the arrow at t = 200 s, 20 μM unlabeled GTPγS was added and the decrease in fluorescence was monitored. The dissociation of BODIPY FL GTPγS was fit with single exponential functions and the half life value was determined i.e. t 1/2 = 9 s suggesting a low affinity for the binding of BODIPY FL GTP. b Monitoring GTP-binding of the P . falciparum G-protein in a time course experiment: Binding assay with different protein concentrations of PfG from Plasmodium green line 62.5 μg, red line 16,5 μg; the reaction was antagonized with unlabeled GTP after 100 s blue line (62,5 μg purified G-protein), purple line (16,5 μg purified-G-protein), yellow line (8,0 μg G-protein).

Article Snippet: Fluorescense exitation (470 nm) and emission (510 nm) were determined in a fluorescense microplate reader (GoMax-Multi, Promega, Karlsruhe).

Techniques: Binding Assay, Fluorescence, Purification

Journal: Nature Communications

Article Title: The mitochondrial ubiquitin ligase MARCH5 resolves MAVS aggregates during antiviral signalling

doi: 10.1038/ncomms8910

Figure Lengend Snippet: ( a ) Immunoblot analysis of MARCH5 expression levels in MARCH5-depleted Raw264.7 cells. See full blots in . ( b ) Twenty-four hours after infection, PR8-GFP or VSV-GFP virus replication assay by fluorescence microscopy (fluorescence, upper; phase-contrast microscopy, bottom) and virus titration by fluorescence analysis or plaque assay in siControl or siMARCH5 expressing Raw264.7 cells. Error bars, mean±s.e.m. ( n =3). Scale bars, 100 μm. ( c ) Determination of the virus titration using fluorescence analysis or plaque assay in March5 +/+ and March5 +/− BMDM cells infected with PR8-GFP or VSV-GFP virus. Error bars, mean±s.e.m. ( n =3). ( d , e ) Bioassay of IFN-β or IL-6 (ELISA) in supernatants of MARCH5-depleted Raw264.7 ( d ) or March5 +/+ and March5 +/− BMDM cells ( e ) infected or transfected with PR8-GFP, VSV-GFP, poly(I:C) or 5′ppp-dsRNA. After cells were transfected with siControl or siMARCH5 for 24 h, followed by infection with virus or transfection with poly(I:C) for indicated times. Error bars, mean±s.e.m. ( n =3). All data are representative of at least three independent experiments.

Article Snippet: GFP-tagged VSV or H1N1 influenza virus (A/PR8/8/34) replication levels were measured with Fluorescence module of GloMax-Multi Microplate Multimode Reader (Promega).

Techniques: Western Blot, Expressing, Infection, Virus, Fluorescence, Microscopy, Titration, Plaque Assay, Bioassay, Enzyme-linked Immunosorbent Assay, Transfection

Journal: Nature Communications

Article Title: The mitochondrial ubiquitin ligase MARCH5 resolves MAVS aggregates during antiviral signalling

doi: 10.1038/ncomms8910

Figure Lengend Snippet: ( a ) After Myc-MARCH5 WT or Myc-MARCH5 H43W expression vectors were introduced into MARCH5 −/− HEK293T cells, cells were infected with PR8-GFP or transfected with poly(I:C) for 24 h. Promoter activity of IFN-β, ISRE, IFN-α or IRF3. Graphs represent fold-induction relative to the luciferase activity in control cells. Error bars, mean±s.e.m. ( n =3). ( b ) Immunoblot analysis of endogenous or ectopic expression levels of MARCH5 in MARCH5 +/+ or MARCH5 −/− HEK293T cells. See full blots in . ( c , d ) Twenty-four hours after infection, VSV-GFP replication assay by fluorescence microscopy analysis ( c ). Bioassay of IFN-β or IL-6 production (ELISA) ( d ). Error bars, mean±s.e.m. ( n =3). Scale bars, 100 μm. ( e , f ) IRES, MARCH5 WT -Flag or MARCH5 H43W -Flag stably expressing Raw264.7 cells were stimulated by infection with VSV-GFP for 24 h. Twenty-four hours after infection, VSV-GFP replication assay by fluorescence microscopy analysis (left). Quantification of GFP intensity or virus titration by plaque assay (right; e ). Bioassay of IFN-β or IL-6 production (ELISA) in supernatants of cultured Raw264.7 stable cells ( f ). Error bars, mean±s.e.m. ( n =3). All data are representative of at least three independent experiments. Scale bars, 100 μm.

Article Snippet: GFP-tagged VSV or H1N1 influenza virus (A/PR8/8/34) replication levels were measured with Fluorescence module of GloMax-Multi Microplate Multimode Reader (Promega).

Techniques: Expressing, Infection, Transfection, Activity Assay, Luciferase, Control, Western Blot, Fluorescence, Microscopy, Bioassay, Enzyme-linked Immunosorbent Assay, Stable Transfection, Virus, Titration, Plaque Assay, Cell Culture