Fig 4. NEC distribution and phosphorylation in transfected HeLa cells.

(A) Potential pUs3 substrates, indicated in red, reside in HSV-2 pUL31 (top) and in HSV-2 pUL34 (bottom). (B) HeLa cells were co-transfected with the expression plasmids indicated on the left of each panel. At 18 hours post transfection, cells were fixed and nuclei were stained with Hoechst 33342. Representative images of cells with bright fluorescence indicative of robust NEC production are shown. Scale bars are 10 μm. (C) To quantify the requirements for extravagation formation, HeLa cells were co-transfected with the plasmids indicated on the x-axis, then fixed and stained as described in (B). Images of 70 to 100 cells were captured for each condition and the percentage of fluorescence outside of the nucleus for each image was determined as described in Materials and Methods. Error bars are standard error of the mean. Asterisks denote significant decreases in the percentage of fluorescence outside of the nucleus in comparison to that of co-transfection with EGFP-pUL31, pUL34 and pUs3, determined by Student’s T test; **** = P value < 0.0001. (D, E) HeLa cells were transfected or co-transfected with the expression plasmid(s) indicated above each lane. Equal volumes of protein extracts prepared at 18 hours post transfection were electrophoresed through regular or phos-tag SDS polyacrylamide gels and proteins transferred to PVDF membranes. Membranes were probed with antisera indicated on the right of each panel. Molecular weight markers in kDa are indicated on the left side of the panels. Black arrowheads denote the position of unphosphorylated protein species.