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Figure 8


Fig. 8. (A) Binding of 14-3-3 to MARK2 and MARK3 after PKC{zeta} co-expression. Wild-type (wt) or indicated mutant forms of GST-MARK isoforms were expressed in HEK 293 cells, in the absence or presence of kinase inactive (D394/A, ki) or wild-type (wt) Flag-PKC{zeta}, purified on glutathione-Sepharose and analysed by western blot with regards to total protein ({alpha}GST) and 14-3-3 binding ({alpha}14-3-3). Expression of Flag-PKC{zeta} was monitored by western blotting of the lysates ({alpha}Flag). (B-E) Subcellular localisation of MARK2 and MARK3 after PKC{zeta} co-expression. (B,D) As in A, except cells were fractionated into a cytosolic and a membrane fraction. Subcellular fractions, including solubilised portions of the homogenates were analysed by western blot with regards to MARK isoforms ({alpha}GST), PKC{zeta} ({alpha}Flag), the cytosolic marker GAPDH ({alpha}GAPDH) and the plasma membrane marker Na-K-ATPase ({alpha}Na-K-ATPase). (C,E) As in A, except that GFP fusions of MARK isoforms were employed and cells were fixed in 3% paraformaldehyde 24 hours post transfection. The cells were subsequently permeabilised and stained with anti-Flag and Alexa Fluor 594-labelled anti-mouse antibody. The fluorescence was analysed by confocal fluorescence microscopy. The cells shown are representative of images obtained in two separate experiments. Bars, 10 µm.