FIG 7.

Apical PM targeting of NA TMD mutants and the effect of cholesterol depletion. Polarized MDCK cells were cotransfected with double (A) and triple (D) combinations of wt HA and NA and mutant expression plasmids, as indicated above the panels. (B) For the cholesterol depletion experiment, polarized MDCK cells were pretreated with lovastatin for 12 h and then cotransfected with wt-HA and wt-NA expression plasmids. After 11 h of incubation at 37°C with lovastatin, the cells were further treated with MβCD for 1 h at 37°C. At 12 hpt, the cells were stained with anti-HA mAb12-1G6 (green) and sheep anti-NA Ab (red). Nuclei were stained with TO-PRO-3 (blue). All images were taken at the same magnification, and images in the x-z planes are shown. Scale bar, 10 μm. For semiquantification, 50 HA and NA double-positive cells (C) and 50 HA, NA, and M1 triple-positive cells (D) were subjected to distribution pattern analysis as described in the legend of Fig. 5.