Fig. 4. (A) Immunofluorescence of 14/Vero cells after heat shock. (A-C) Heat-shocked cells were detected for Hsp70 (A) and UL14 protein (B), showing that the two proteins colocalize in the nucleus and nucleolus (C, merge). (D-F) 14/Vero cells were microinjected with anti-Hsp70 polyclonal antibody and treated with heat shock. The cells were detected for the injected antibody (D) and UL14 protein (E; F, merge). UL14 protein still translocated into the nucleus and nucleolus in microinjected cells, indicating that its localization did not depend on Hsp70 function. (B) The comparison of nucleolar localization of UL14 protein and Hsp70 in Vero and 14/Vero cells. Vero and 14/Vero cells were heat-shocked at 43°C for 120 minutes and recovered at 37°C for up to 16 hours. Cells were fixed at several time points for immunofluorecence. Bright nucleolar staining of Hsp70 and/or UL14 protein was counted and the population was plotted against time. In both cells, Hsp70 rapidly translocated to the nucleoli after heat shock and started to delocalize to the cytoplasm during recovery. The rate of delocalization was faster in 14/Vero cells than in Vero cells. In 14/Vero cells, UL14 protein remained in the nucleoli for a longer time than Hsp70. (C) Hsc70/Hsp70 expression in heat-shocked Vero and 14/Vero cells. Cells were continuously heat-shocked at 43°C for up to 60 minutes and samples were collected for immunoblotting at the intervals shown. Anti-Hsp70 Ab was used at a dilution of 1/5000. To show that nearly equal amounts of protein were loaded, bands corresponding to actin were obtained from staining identical SDS-PAGE gels with CBB.