Journal: Virology

Article Title: Porcine reproductive and respiratory syndrome virus induces HMGB1 secretion via activating PKC-delta to trigger inflammatory response.

doi: 10.1016/j.virol.2018.02.021

Figure Lengend Snippet: Fig. 7. PRRSV ORF2b and ORF5a products (E and pORF5a, respectively) are responsible for inducing HMGB1 secretion via activating PKCδ. A. Screening of PRRSV ORFs. HEK293 cells were transfected with individual GFP-ORF plasmids. The SUP and WCL were harvested 30 h after transfection for WB with antibodies against HMGB1, tubulin and GFP. Empty GFP vector (EV) was included as a control in the transient transfection. The expression of ORF2 and ORF3 were also confirmed by fluorescence microscopy shown in the right panel of GFP images. Bars in the images denote 50 µm. B. ORF2b and ORF5a products induce HMGB1 secretion in a dose-dependent manner. HEK293 cells were transfected with incremental amounts of GFP- ORF2b or GFP-ORF5a plasmid, respectively. At 30 h after transfection, the SUP and WCL were harvested for WB. Relative levels of HMGB1 in SUP are shown as folds below the images in densitometry analysis. C. ORF2b and ORF5a products activate PKCδ. HEK293 cells were transfected with EV, ORF2b, ORF5 or ORF5a plasmid and 30 h later, harvested for WB with antibodies against PKCδ-S645, PKCδ, tubulin and GFP. Relative levels of PKCδ-S645 are shown as folds below the images after normalization with tubulin. D. ORF2b and ORF5a products induce PKCδ activation in a dose-dependent manner. HEK293 cells were transfected with incremental amounts of ORF2b or ORF5a plasmid and 30 h after transfection, harvested for WB. Relative levels of PKCδ-S645 are shown as folds below the images after normalization with tubulin.

Article Snippet: Fluorescence signal was observed under confocal fluorescence microscopy (Nikon C2) and images were taken with NISElements version 4.0 (Nikon).

Techniques: Transfection, Plasmid Preparation, Control, Expressing, Microscopy, Activation Assay