Journal: Virology

Article Title: Tetherin restricts HSV-2 release and is counteracted by multiple viral glycoproteins.

doi: 10.1016/j.virol.2014.11.005

Figure Lengend Snippet: Fig. 4. Tetherin is highly likely to be incorporated into HSV-2 virions. (A) HSV-2 virion preparations were fractioned via a sucrose gradient cushion (10–50%) to separate cellular membrane fragments and defective viral particles from intact infectious virions. The representative viral and cellular proteins were assessed by western blot. One representative experiment out of three is shown. (B) Immunofluorescence analysis of HSV-2 virions. Virions harvested from HSV-2 infected HeLa cells were pelleted and applied to poly-L-lysine-coated coverslips prior to immunofluorescence analysis using anti-ICP5 and anti-tetherin antibodies. The FITC-positive dots were positive for tetherin and the Cy3-positive dots were positive for HSV-2 ICP5. Representative fields observed in two experiments are shown. Scale bars represent 500 nm.

Article Snippet: Cells were incubated for 1 h at 37 1C with a mouse monoclonal antibody against FLAG (F1804; Sigma) at a dilution of 1:200, gB (mouse monoclonal antibody; ab6506; Abcam) at a dilution of 1:200, gD (mouse monoclonal antibody; sc-58154; Santa Cruz) at a dilution of 1:50 or HSV-2 (sheep polyclonal antibody; PAB13979; Abnova) at a dilution of 1:200, followed by an incubation for 1 h at 37 1C with a FITC-conjugated goat anti-mouse secondary antibody (Boster, China) at a dilution of 1:100, a Cy3-conjugated goat antirabbit secondary antibody (Boster, China) at a dilution of 1:100 or a FITC-conjugated rabbit anti-goat secondary antibody (Boster, China) at a dilution of 1:100 in PBS-2% (w/v) BSA.

Techniques: Membrane, Western Blot, Infection

Journal: PLoS ONE

Article Title: Altered Protein Networks and Cellular Pathways in Severe West Nile Disease in Mice

doi: 10.1371/journal.pone.0068318

Figure Lengend Snippet: (A) Protein samples from each group used for proteomic analysis were minimally labeled with cyanine-3 dye. At the top, a representative protein profile of three biological replicates from brain lysates of mock-, and WNV early- and late-infected mice, separated by 10% SDS-PAGE is shown. WB with fluorescence-based methods was used to detect an overlaid fluorescent scan of the general protein patterns (Cy3 dye (green)) and the specific immunoreactive proteins (FITC or Cy5 dye (red)). To better visualize protein detection signals observed with each specific antibody used, corresponding cropped WB images are presented in grey levels. (B) The graphs correspond to the mean ± S.D. of protein quantity measured by densitometry of the antigenic bands. Densitometry analyses were performed using TotalLab Quant v12.2 software (Nonlinear Dynamics), and data were normalized to levels of global protein pattern intensity. The values indicated under each graph correspond to fold changes from paired comparisons ( i.e., WNV-E/mock, WNV-L/mock, WNV-L/WNV-E). WNV-E/−L, biological replicates of brain samples infected by West Nile Virus and collected at early or late time-points. The significance of the differential protein expression are indicated *, p<0.05; **, p<0.01; ***, p<0.001. A.U., arbitrary units. α-, antibody anti-; ★, IFN-γ activated mice bone marrow derived macrophage (p-701-STAT1 positive control); #, no quantification for p-701-STAT1. CAPN9, calpain 9; CLTC, clathrin heavy chain; DNM1, dynamin 1; GFAP, glial fibrillary acidic protein; HUWE1; E3 ubiquitin-protein ligase; MAP1B, microtubule-associated protein 1B; MAP2, microtubule-associated protein 2; PRDX6, peroxiredoxin 6; STAT1/2, signal transducer and activator of transcription 1/2; VIM, vimentin.

Article Snippet: After three washes in PBS-T, primary rabbit antibodies were revealed with ECL Plex goat anti-rabbit IgG Cy5-conjugated secondary antibody (1∶1000, GE Healthcare), and rabbit anti-goat IgG fluorescein isothiocyanate (FITC) conjugate (1∶400, Southern Biotech, Birmingham, AL) was used for the detection of anti-CAPN9 goat antibody diluted in PBS-T-milk.

Techniques: Labeling, Infection, SDS Page, Fluorescence, Software, Virus, Expressing, Derivative Assay, Positive Control, Ubiquitin Proteomics