Journal: Journal of Virology

Article Title: Impact of the changes in substrate specificity of herpes simplex virus 1 protein kinase Us3 on viral infection in vitro and in vivo

doi: 10.1128/jvi.00400-25

Figure Lengend Snippet: Effects of the PKA inhibitor on the phosphorylation levels of proteins detected by the 100G7E antibody. ( A and B ) Vero cells were mock-infected ( A ) or infected with wild-type HSV-1(F) ( B ) at an MOI of 10, treated with 2 µM staurosporine (STS) or DMSO 12 h post-infection, harvested 18 h post-infection, and analyzed by immunoblotting using the SimpleWestern capillary system with antibodies to anti-phospho-PKA substrate 100G7E or β-actin. Digital images are representative of three ( A ) or four ( B ) independent experiments. A molecular mass marker is indicated on the left. A high-intensity image is also shown (right panel of A). (C to H) The accumulation levels corresponding to p143 ( C ), p79 ( D ), p61 ( E ), p49 ( F ), p37 ( G ), and p34 ( H ) in the experiments shown in panel B were quantified and normalized to those of β-actin. Values represent the mean ± SEM of four independent experiments, expressed relative to the values obtained from DMSO-treated HSV-1(F)-infected Vero cells, which were normalized to 100%. Statistical significance was assessed by an unpaired two-tailed Student t -test: * ; P < 0.05, n.s., not significant.

Article Snippet: The PKA inhibitor staurosporine (STS) was purchased from Selleck Chemicals (Texas, USA) and was used at a final concentration of 2 μM.

Techniques: Phospho-proteomics, Infection, Western Blot, Marker, Two Tailed Test

Journal: Journal of Virology

Article Title: Epizootic Hemorrhagic Disease Virus Induces and Benefits from Cell Stress, Autophagy, and Apoptosis

doi: 10.1128/JVI.02116-13

Figure Lengend Snippet: EHDV2-IBA induces the apoptosis of OK cells. (A and B) Subconfluent cultures of OK cells, at 24 h postplating, were either noninfected or infected for the indicated times and left untreated or treated with staurosporine (STS; 1 μM for 20 h). Cells were collected from the medium or suspended by trypsinization and processed for FACS analysis. (A) The graphs depict the FACS measurements of DNA content of propidium iodide-stained OK cells: 1, sub-G1; 2, G0/G1; 3, S; 4,G2/M. (B) The graph shows the average ± SE of the percentage of cells presenting sub-G1 DNA content under the indicated experimental conditions (n = 3; *, P < 0.05; **, P < 0.01). (C) OK cells at 24 h postplating were noninfected or infected with EHDV2-IBA. At indicated times points, cells were collected and processed for 10% SDS-PAGE (see Materials and Methods). The panel depicts a typical immunoblot probed with the anti-NS3, anti-caspase 3, and anti-MEK (loading control) antibodies. tMEK, total MEK (MEK1/2). (D) Cells cultured and infected as described for panel C were processed for DNA laddering (see Materials and Methods) at the indicated time points. The panel depicts a representative DNA laddering experiment (n = 5) employing equal amounts of genomic DNA, extracted from cells under the indicated conditions, and separated on a 2% agarose gel. (E) OK cells, cultured as described for panel A and either noninfected or infected with UV-irradiated EHDV2-IBA for 72 h, were processed for FACS analysis of DNA content. Graphs depict typical FACS measurements of cells infected with EHDV2-IBA (left) or UV-irradiated EHDV2-IBA (right). (F) Representative agarose gel from a DNA laddering experiment of OK cells noninfected or infected with EHDV2-IBA or UV-irradiated EHDV2-IBA (60 hpi) and left untreated or treated with STS (1 μM for 20 h).

Article Snippet: Reagents were employed at the following final concentrations: staurosporine (STS; A.G. Scientific Inc., San Diego, CA, USA), 1 μM; caspase inhibitor Q-VD-OPH (quinolyl-valyl-O-methylaspartyl-[-2,6-difluorophenoxy]-methyl ketone; Kamiya Biomedical, Seattle, WA, USA), 20 μM; 3-methyladenine (3MA; Sigma-Aldrich, St. Louis, MO, USA), 10 mM; JNK inhibitor SP600125 (Merck KGaA, Darmstadt, Germany), 20 μM; roscovitine (Sigma-Aldrich, St. Louis, MO, USA), 20, 40, or 80 μM.

Techniques: Infection, Staining, SDS Page, Western Blot, Cell Culture, DNA Laddering, Agarose Gel Electrophoresis, Irradiation