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Fig. 2. Insulin stimulates PIKfyve serine318 phosphorylation in intact cells. (A) Cells transiently transfected with wild-type GFP-PIKfyve (WT), the GFP PIKfyve[S318A] mutant or vector alone (V) were pre-incubated in the absence (C) or presence of wortmannin (W) for 30 minutes and then in the absence or presence of insulin (I) for a further 15 minutes. Cell lysates were then subjected to western blotting with either the anti-pS318 or anti-PIKfyve antibodies as indicated. Note that the anti-pS318 antibody crossreacts with a phosphoprotein that migrates just below PIKfyve and exhibits an increase in phosphorylation in the presence of wortmannin. The identity of this phosphoprotein is unknown but it is not endogenous PIKfyve as demonstrated by the lack of reactivity with an anti-PIKfyve antibody (lower panel). (B) Wild-type GFP-PIKfyve or a GFP-PIKfyve[R192A] mutant were transiently transfected into CHO.T cells. 24 hours later the cells were examined by confocal microscopy. Typical images of the distribution of these proteins are shown. Bar, 10 µm. (C) Cells were transfected with wild-type GFP-PIKfyve or the GFP-PIKfyve[R192A] mutant and treated as described in panel A for western blotting with either the anti-pS318 or anti-PIKfyve antibodies. (D) Data from at least three experiments performed according to panel C were quantified by densitometric scanning of the autoradiographs. The data are corrected for the amount of GFP-PIKfyve loaded onto the gels and are expressed relative to the appropriate control.
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