Figure 7
Mapping Ad5 L4-100K binding regions in chimeric hexons and effect of Ad5 L4-100K on hexon trimerization.
(a) Schematic strategy to construct truncated hexons of 5-Hexon hypervariable region (5-HVR) and 5-Hexon conserved region (5-CR). (b) HEK293 cells (1 × 106) were co-transfected with 2 μg HA-tagged Ad5 L4-100K and 2 μg 5-HVR or 5-CR. Cells were co-immunoprecipitated with HA beads followed by SDS-PAGE and immunoblot analysis using anti-HA and anti-hexon antibodies. (c) A schematic strategy to construct truncated Ad5 L4-100K. (d) HEK293 cells (1 × 106) were co-transfected with 6 μg truncated L4-100K with HA-tag and 6 μg 5-Hexon. Cells were co-immunoprecipitated with HA beads followed by SDS-PAGE and immunoblot analysis using anti-HA and anti-hexon antibodies. All co-immunoprecipitation experiments were repeated three times. (e) HEK293 cells were transfected with Ad5 L4-100K, Ta, Tb, Tc, Td and 5-Hexon. Cells were harvested at 72 h. Total cell extracts were prepared and hexon trimers were separated by native-PAGE and subjected to immunoblotting using an anti-hexon goat pAb and mouse tubulin mAb as a loading control.
