Fig. 1.

DRG neurons transduced by p.M_SΔAkt and p.M_FΔAkt display different subcellular targeting of the Akt/EGFP fusion protein. A: Western blot detection of HA-epitope tag or phospho-Akt_S473 (p-Akt) of neuronal cultures transduced with M_SΔAkt or M_FΔAkt revealed efficient and similar levels of expression of the myristoylated transgene (ΔAkt). Addition of NGF (positive control) increased the levels of p-Akt, whereas cultures transduced with p.EGFP had levels of p-Akt similar levels in to control cultures. B: Peroxidase activity from CTB-HRP was detected by measuring the level of oxidized ABTS, which absorbs light at 420 nm. Neuronal cultures lysed at 0°C had high levels of peroxidase activity in fraction 4, indicating the presence of buoyant lipid rafts. Incubation of neuronal cultures in Triton X-100 at 37°C, known to disrupt lipid rafts, abolishes the peak in peroxidase activity in fraction 4. C: Detection of EGFP (Em₅₀₈) from sucrose gradient fractions indicated lipid raft accumulation of M_FΔAkt. In cultures incubated on ice, which maintains the integrity of the lipid rafts, greater amounts of EGFP in the lipid raft fraction (fraction 4) of neuronal cultures that expressed the M_FΔAkt fusion protein were detected compared with cultures that expressed the M_SΔAkt fusion protein. Disruption of lipid rafts with Triton X-100 at 37°C before fractionation decreased the amount of Akt/EGFP fusion protein in fraction 4 of neurons that expressed M_FΔAkt fusion protein to control levels.