Fig. 1. Overexpression of PKC in MDCK cells induces the lamellipodia. (A) The expression of endogenous PKC and GFP-PKC was analyzed by immunoblotting with anti-PKC (top panel). The activity of GFP-PKC or its kd mutant was analyzed by an in vitro kinase assay using myelin basic protein (MBP) as an exogenous substrate (bottom panel). (B) Total PKC (including endogenous PKC and GFP-PKC) was immunoprecipitated by polyclonal anti-PKC and its activity was analyzed by an in vitro kinase assay. The phosphorylation of MBP was measured and expressed as fold increase relative to the level of the control MDCK cells. The value (mean ± s.d.) was from three experiments. ^*P<0.05. (C) The activity of GFP-PKC was measured in the presence of PMA (150 nM) or rottlerin (5 µM). The phosphorylation of MBP was measured and expressed as fold increase relative to the level in the absence of PMA and rottlerin. The value (mean ± s.d.) was from three experiments. ^*P<0.05. (D) MDCK cells stably expressing GFP, GFP-PKC or its kd mutant were allowed to grow as cell colonies and the micrographs were taken under a phase-contrast microscope. Bar, 50 µm. (E) MDCK cells were plated on collagen-coated glass coverslips for 24 hours and stained for actin filaments with TRITC-conjugated phalloidin. The fluorescence of GFP and the actin filaments was visualized under a confocal laser-scanning microscope. Arrowheads indicate the distribution of GFP-PKC and the cortical actin filaments at the edge of membrane protrusions. Bars, 20 µm. (F) An equal amount of the whole cell lysates from MDCK cells stably expressing FLAG-PKC or their neomycin-resistant control cells (Neo) was analyzed by immunoblotting with anti-FLAG or anti-PKC. (G) MDCK cells as described in F were plated on collagen-coated glass coverslips for 24 hours and stained for FLAG-PKC with anti-FLAG. Bar, 20 µm.