Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: VIPER is a genetically encoded peptide tag for fluorescence and electron microscopy

doi: 10.1073/pnas.1808626115

Figure Lengend Snippet: VIPER-tagged transferrin receptor retains transferrin binding and endocytosis. (A) CHO TRVb cells expressing TfR1-CoilE were treated with CoilR-Cy5 and fluorescent ligand (Tf-AF488). In live cells, labeling by both Tf-AF488 and CoilR-Cy5 was localized to the cell surface at 0 min. After 30 min, AF488 and Cy5 signals from the Tf-TfR1 complex were observed together in endocytic vesicles. (B) Cells expressing TfR1-mCherry were treated with Tf-AF488. In A and B, yellow boxes delineate Insets, which provide a 2× magnified view. The merged images (Right column) include Tf-AF488 (green), nuclear stain (blue), and either mCherry (magenta) or CoilR-Cy5 (magenta). (Scale bars: 25 μm.)

Article Snippet: For comparative analysis, we acquired a vector encoding TfR1 fused to the monomeric red fluorescent protein mCherry (pcDNA3_TfR1-mCherry; Addgene #55144).

Techniques: Binding Assay, Expressing, Labeling, Staining

Journal: Nature Communications

Article Title: Plasma-derived extracellular vesicles from Plasmodium vivax patients signal spleen fibroblasts via NF-kB facilitating parasite cytoadherence

doi: 10.1038/s41467-020-16337-y

Figure Lengend Snippet: a BCA (protein concentration) of circulating EVs from healthy donors ( h EVs) and P. vivax patients ( Pv EVs) ( n = 10, individual samples). Data show mean ± SD. Two-sided, Mann–Whitney test, * p = 0.0232 (GraphPad). b P. vivax proteins identified by different unique peptides (sequences below the description of corresponding proteins). UniProtKB accession numbers and gene name corresponding to the P. vivax PvP01 strain are shown. c Western blot analysis of Pv EVs obtained from different individual patients (Pv1-7 and Pv10) and human donors (H1 and H3) using anti P. vivax MSP3.1 (upper membrane) and PHISTc (bottom membrane) antibodies. MSP3.1 and PHISTc recombinant truncated-proteins fused to GST, were used as positive controls. Molecular weight in kDaltons (kDa) is shown to the left. M: molecular size marker. Image representative of three independent experiments. d Nanoparticle tracking analysis (NTA) profile (size [nm] versus concentration [particles/mL]) of pooled h EVs and Pv EVs was analysed using NanoSight LM10-12. Table shows mean of three measurement of pooled h EVs and Pv EVs quantified by NTA (particles concentration) and BCA (protein concentration). e Beads-based flow cytometry analysis of pooled h EVs and Pv EVs using six EV markers (CD9, CD63, CD81, GAL3, CD5L and CD71). Data show median fluorescence intensity (MFI) of each antibody and control antibodies (rabbit and mouse-isotype) ± SD (technical replicates, n = 3). Unpaired and two-sided, t -test ** p = 0.0032 (CD63), *** p = 0.0006 (CD81, CD71), **** p < 0.0001 (CD9) (GraphPad). Source data are provided as a Source data file and Supplementary Data , and .

Article Snippet: Further, cells were stained using the following antibodies: CD90-PE/Cy7 [5E10] (Biolegend, Cat#328124) 1/100; CD44-FITC [KM201] (Abcam Cat#ab25340) 1/100; CD54-Alexa Fluor® 488 [HCD54] (Biolegend, Cat#322714) 1/400; CD71-PE [AC102] (Miltenyi Biotec, Cat#130-091-728) 1/400; CD45-PerCP [2D1] (BD Biosciences, Cat#345809) 1/50.

Techniques: Protein Concentration, MANN-WHITNEY, Western Blot, Membrane, Recombinant, Molecular Weight, Marker, Concentration Assay, Flow Cytometry, Fluorescence, Control