Fig. 6. PKC-mediated phosphorylation of adducin promotes cell spreading and migration. (A) GFP, GFP--adducin or GFP--adducin (or its S726A mutant) was expressed alone or co-expressed with FLAG-PKC in MDCK cells. An equal amount of the whole cell lysates from those cells was analyzed by immunoblotting with anti-adducin, anti-phospho-adducin or anti-FLAG. (B) MDCK cells stably co-expressing FLAG-PKC with GFP--adducin were fixed and stained with anti-FLAG. The fluorescent images of FLAG-PKC and GFP--adducin were scanned by a confocal microscope on the z-axis 2 µm above the substratum. Bar, 5 µm. The enlarged images show the colocalization of GFP--adducin and FLAG-PKC at the plasma membrane. (C) MDCK cells were suspended in serum-free medium and plated on collagen-coated dishes. 40 minutes later, spread cells were counted and expressed as the percentage in total counted cells (n=200). Values (means ± s.d.) are from three independent experiments. (D) The cells were suspended in serum-free medium and subjected to a Trans-well cell migration assay. 6 hours later, the migrated cells were fixed, stained and counted using a light microscope. Values (means ± s.d.) are from three independent experiments and expressed as fold increase relative to the level of the cells expressing GFP alone. ^*P<0.05 (compared with the cells expressing GFP alone). ^**P<0.05 (compared with the cells co-expressing FLAG-PKC and GFP).