Figure 6.

14-3-3 is sufficient and required for CCTα nuclear import. A) MLE cells were plated on 35-mm glass-bottom tissue culture dishes at 100,000 cells/dish, transfected with YFP-14-3-3 or V5-tagged R18 (2 μg/dish) using Fugene 6. At 24 h after transfection, cells were treated with or without calcium chloride (1.0 mM) in serum-free DMEM/F12 at 37°C for 1 h. Cells were fixed and immunostained with an antibody recognizing CCTα, followed by Alexa 488 fluorescent probes; cells were also counterstained with DAPI (to visualize the nucleus). Scale bar = 10 μm. B) Fluorescent signals within the cytosol and the nucleus were calculated and graphed and expressed as ratios of nuclear/cytoplasmic intensity. Insets: immunoblotting was performed to confirm overexpressed 14-3-3 (left) and R18 levels (right) vs. untransfected cells [control (C)], the latter detected using V5 antibodies.