Fig. 4. The binding of 14-3-3 proteins to serine 294 does not regulate nucleo-cytoplasmic shuttling of PI4KIIIß. (A) COS7 cells were transfected with wildtype Flag-PI4KIIIß or Flag-PI4KIIIß_S294A. Cells were incubated with the vehicle (0.1% ethanol, control) or treated for 16 hours with 10 ng/ml leptomycin B. After incubation cells were fixed and stained with anti-Flag followed by anti-mouse IgG Alexa488. (B) COS7 cells were transfected with either Flag-PI4KIIIß alone (upper panel) or together with DN-HA-14-3-3 (lower panel). After 24 hours' incubation, cells were fixed and stained with anti-Flag followed by anti-mouse IgG Alexa488 and anti-14-3-3 followed by anti-rabbit IgG Alexa546. Flag-PI4KIIIß is stained in green, 14-3-3 proteins in red. Arrowheads indicate double-transfected cells. A series of images was taken at 0.5-µm intervals through the Z plane of the cell and were processed to form a projected image. Bar, 10 µm.