Journal: Journal of Biological Chemistry

Article Title: Proteomic Analysis of DC-SIGN on Dendritic Cells Detects Tetramers Required for Ligand Binding but No Association with CD4

doi: 10.1074/jbc.m402741200

Figure Lengend Snippet: FIG. 6. FRET measurements between CD4 and DC-SIGN on iMDDCs and between CD4 and CD71 on CemT4 cells and iMDDCs. Energy transfer efficiencies were measured using different fluorescein conjugated antibodies against CD4 (OKT4 and Q4120) as donor antibodies and Cy3-conjugated antibodies against DC-SIGN, CD4 (OKT4) and the TFR (WM82). Energy transfer efficiencies were calculated as the percentage of donor fluorescence quenching in the presence of an acceptor antibody and were measured either before (mock) stimulation (dark bars) and after 15 min incubation with or without a stimulus (shaded bars). Shown are the mean energy transfer efficiencies of ten independent experiments (OKT4/DC-SIGN) or four independent experiments (all others). A significant energy transfer (5% to 7%) can only be detected on CemT4 cells between CD4 and the TFR after PMA stimulation. No significant transfer was detected for this interaction on iMDDCs and between CD4 and DC-SIGN on iMDDCs.

Article Snippet: Disuccinimidyl suberate (DSS), dithiobis(succinimidylpropionate) (DSP), were from Pierce, protein G-Sepharose and Cy3 monoreactive NHS ester from Amersham Biosciences.

Techniques: Fluorescence, Incubation

Journal: Nature Communications

Article Title: Emergent mechanics of a networked multivalent protein condensate

doi: 10.1038/s41467-025-60345-9

Figure Lengend Snippet: a Images of differential interference contrast (DIC, left) and fluorescence (right) of the 6xPSD droplets with 1% PSD-95 labeled by a fluorescent dye Cy3. Scale bar: 20 μm. b Six example sets of time-lapse images of FRAP obtained from individual 6xPSD droplets with 1% single protein component being labeled by Cy3. Scale bar: 5 μm. c Normalized FRAP mean intensity I ( t ) of the 6xPSD droplets as a function of time t before ( t < 0) and after ( t > 0) photo-bleaching. The mean value I ( t ) is obtained by averaging the FRAP intensity over 8 droplets for PSD-95, 4 droplets for GKAP, 9 droplets for Shank3, 8 droplets for Homer3, 8 droplets for NR2B and 10 droplets for SynGAP. The error bars show the standard deviations of the measurements. The solid lines are the best fits of Eq. to the FRAP data. d Two-dimensional (2D) fluorescence intensity maps obtained by 3D confocal scanning of a 6xPSD droplet with 1% Shank3 labeled by Cy3 (upper panel). Scale bar: 5 μm. A 1D intensity profile across the diameter of a horizontal section (XY plane) of the droplet at its brightest section (bottom panel). a.u. arbitrary units. Similar images were obtained in multiple experiments ( N ≥ 3). e Fluorescence intensity images obtained from a solution of the protein Shank3 labeled by Cy3 with three different concentrations: 5 μM, 10 μM, and 20 μM (upper panel, scale bar: 5 μm) and the corresponding 1D intensity profiles (bottom panel). a.u. arbitrary units. Similar images were obtained in multiple experiments ( N ≥ 3). f Three repeated runs of the centrifuge assay , in which a high-speed centrifuge is used to separate the dilute phase (Supernatant, S) and condensed phase (Pellet, P). Protein components in each phase are further separated by electrophoresis (SDS-PAGE). The band intensities in the gel are used to quantify the pellet percentage and supernatant percentage of each protein component (the sum of the two percentages is unity). The NR2B band is quantified by the gel with sliver staining (see Supplementary Fig. 5). g Obtained pellet percentage of the six protein components from N = 3 measurements. Source data are provided as a Source Data file.

Article Snippet: Cy3 NHS ester dye (AAT Bioquest) is dissolved by DMSO and incubated with the corresponding protein at room temperature for 2 h (fluorophore to protein molar ratio is 1:1).

Techniques: Fluorescence, Labeling, Electrophoresis, SDS Page, Staining

Journal: Neuron

Article Title: Phase Separation-mediated TARP/MAGUK Complex Condensation and AMPA Receptor Synaptic Transmission

doi: 10.1016/j.neuron.2019.08.001

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Alexa 488 NHS ester (ThermoFisher), Alexa 647 NHS ester (Invitrogen) and Cy3/iFluor 405 NHS ester (AAT Bioquest), were dissolved by DMSO and incubated with the corresponding protein at room temperature for 1 hour (fluorophore to protein molar ratio was 1:1).

Techniques: Recombinant, Mutagenesis, Plasmid Preparation, Expressing, Sequencing, Software

Journal: Parasites & Vectors

Article Title: Fasciola gigantica excretory-secretory products (FgESPs) modulate the differentiation and immune functions of buffalo dendritic cells through a mechanism involving DNMT1 and TET1

doi: 10.1186/s13071-020-04220-0

Figure Lengend Snippet: FgESPs do not directly interact with DNMT1 or TET1 in buffalo DCs. a Western blot analysis for the Co-IP assay to determine the interaction between FgESPs and DNMT1 or TET1 protein in buffalo DCs. Lanes 1–3: protein-G beads-eluted ‘Ag-Ab-unknown interactive protein’ complex protein sample specifically bounded to anti-DNMT1 mAb (Lane 1), anti-TET1 mAb (Lane 2) or normal mouse IgG (Lane 3) as control Ab from FgESPs-treated DCs; Lanes 4–6: protein-G beads-eluted ‘Ag-Ab-unknown interactive protein’ complex protein sample specifically bounded to anti-DNMT1 mAb (Lane 4), anti-TET1 mAb (Lane 5) or normal mouse IgG (Lane 6) from untreated DCs. The laboratory-made FgESPs-specific pAb was used as a primary antibody for all lanes. The immunoblotting result showed that the band pattern among all protein samples had no significant difference. b Fluorescent subcellular localization for FgESPs in buffalo DCs observed under a fluorescence inverted microscope. DAPI (blue) stained the cell nucleus; lipophilic dye DiO (green) strongly bound to the cell membrane; and Cy3-pre-labelled FgESPs emitted orange fluorescence. The merged image demonstrated that FgESPs might not locate in the nucleus. Scale-bars : 20 μm

Article Snippet: The total protein of FgESPs was pre-stained using a fluorescent Cy3 NHS ester (ApexBio Technology, Houston, TX, USA).

Techniques: Western Blot, Co-Immunoprecipitation Assay, Control, Fluorescence, Inverted Microscopy, Staining, Membrane