FIG. 2.

Ras proteins with a palmitoylated carboxy terminus localize to low-density raft fractions and accumulate in the Golgi apparatus. (A) Sonicates of Ba/F3-Fms cells expressing the indicated myc-tagged Ras constructs were fractionated by buoyant density using sucrose gradients. Twelve fractions were collected from the top of the gradient. Fractions 1 and 2 were discarded. After dilution, membranes were pelleted by further ultracentrifugation. The pellets were resuspended in SDS loading buffer and subjected to SDS-PAGE and blotting with anti-myc antibodies to visualize Ras proteins. The top blot indicates the location of Lyn, a marker for rafts. Each blot is representative of at least three independent experiments. (B) NIH 3T3 cells were transiently transfected with GFP-tagged Ras constructs as indicated. The cells were stained with a red fluorescent ceramide (Molecular Probes) to mark the localization of the Golgi apparatus, fixed, and imaged by confocal microscopy. Merged images are shown. The yellow color seen in the case of Ras proteins with palmitoylated carboxy termini indicates the colocalization of green (Ras protein) and red (Golgi marker). Box size, 63 by 63 μm.