Figure 5
Leptin and insulin increase PtdIns(3,4,5)P 3 in isolated neurones Acutely isolated ARC neurones were incubated in the absence and presence of 10 nM leptin (A) or 1 nM insulin (B) for 10 minutes. Cells were fixed and permeabilized, as described in Methods, prior to incubation with wild type (wt) or K273A mutant (mt) PH-GRP1-GFP fusion protein for 1 hour. Cells were subsequently processed for visualising GFP by confocal microscopy. Note that leptin and insulin increased the binding of wild type PH-GRP1-GFP in ARC neurones, and this is shown as both the fluorescence image (upper panels in A, B) and as a false colour image (lower panels in A, B), where blue represents low or non-detectable fluorescence and red the highest fluorescence intensity.