Figure 4.

Analysis of Untagged EN-containing ORF2 Fragments: Selected untagged EN-containing ORF2 fragments behaved similarly to the corresponding VP16- and Gal4-tagged EN-containing ORF2 fragments in regards to cytotoxicity and subcellular distribution. (A) Schematic of untagged ORF2 fragments used in this experiment with ORF2 displayed above for reference. (B) Chronic toxicity of EN-containing ORF2 fragments. All ORF2 fragments were cytotoxic as compared to their nonfunctional controls. Error bars denote standard deviation (n = 3). Statistical significance assessed using Student's t-test (*p < 0.05). (C) Analysis of toxicity grouping of EN-containing ORF2 fragments. Analysis is identical to Figure 2B and 2C (*p < 0.05). (D) Western blot analysis of HeLa cells transiently transfected with indicated constructs using anti-ORF2p monoclonal antibodies. Both whole cell lysate and cytoplasmic and nuclear cell fractions were analyzed. Highest molecular weight band in each sample corresponds to expected size of each construct, with larger EN-containing fragments undergoing processing to yield smaller products (*: EN347Δ and ENZ490Δ). Control (C) is cells transfected with empty vector. Cellular fraction is indicated on the right. Molecular weights are indicated on the left. GAPDH (total and cytoplasmic (cyto)) and Lamin A/C (L A/C: nuclear (nuc)) used as loading control. Relative distribution in subcellular compartments of EN269Δ and EN274Δ is quantified in (E). (E) Quantitation of relative distribution of EN-containing ORF2 fragments. Signal intensity of EN-containing ORF2p fragments in each compartment was normalized to loading control. Normalized signal was summed for each cellular compartment and proportion protein expression quantitated per compartment (Example for Cytoplasmic: Normalized Cytoplasmic Signal/(Normalized Cytoplasmic Signal+Normalized Nuclear Signal)). Error bars denote standard deviation (n = 3). Statistical significance was assessed using Student's t-test (*p < 0.05).