Prenylated N-Ras is largely cytosolic. (A) Jurkat T cells were transfected with GFP-tagged N-, H-, or K-Ras4B and imaged alive with a laser-scanning confocal microscope. N, nucleus. Arrowheads indicate Golgi, and the asterisk indicates cytosol. Bar, 10 µm. (B) S350 (S) and P350 (P) fractions were prepared from MDCK cells expressing the indicated GFP-tagged Ras protein that were metabolically labeled with [³⁵S]methionine/cysteine for 17 h. GFP-tagged Ras proteins were immunoprecipitated from detergent extracts of the two fractions. Protein levels were analyzed by phosphorimager and the cytosolic fractions plotted as percentages of total Ras; mean ± SEM, n = 3. A representative radiograph is shown as an inset. (C) The indicated cell types were fractionated as in B. Cell equivalents from each fraction were loaded and analyzed for the indicated proteins by immunoblot. Epidermal growth factor receptor (EGFR) and RhoGDI serve as markers for membrane and cytosol, respectively. Representative data are shown (n = 3). (D) S350 and P350 fractions were generated from MDCK cells stably expressing GFP–N-Ras or GFP–K-Ras4B at endogenous levels that were metabolically labeled with [³⁵S]methionine/cysteine, [³H]melavonate, or [¹²⁵I]iodopalmitate, and immunoprecipitated Ras was analyzed by fluorimetry (³H) or phosphorimager (³⁵S and ¹²⁵I).