Fig. 3. (A) Fractionation of 14/HEp-2 cells during continuous heat shock at 43°C. Cells were heat-shocked for up to 180 minutes, and fractionated into insoluble (pellet, P) and soluble (supernatant, S) fractions at 0, 5, 15, 60, 120 and 180 minutes with PBS containing 1% Triton X-100. The samples were separated by SDS-PAGE, subjected to western blotting and finally detected with anti-UL14 polyclonal antibody. The total amount of the protein decreased after 60 minutes of heat shock, but the amount of UL14 protein in the insoluble fractions was sustained. (B) The nucleolar localization of UL14 protein in 14/Vero cells during heat shock at 43°C and recovery at 37°C. Cells were heat-shocked at 43°C for 120 minutes and left to recover at 37°C for up to 23 hours. Cells were fixed at several time points for immunofluorecence. Bright nucleolar staining of UL14 protein was counted and the population was plotted against time. Nearly all of the cells exhibited nucleolar localization of UL14 protein after 10 minutes of heat shock. The protein gradually delocalized from the nucleolus and, subsequently, from the nucleus during recovery at 37°C.