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. 2007 Jul 23;27(19):6647–6658. doi: 10.1128/MCB.00155-07

FIG. 8.

FIG. 8.

RasV12 acutely downregulates Rap1GAP protein and message levels. (A) WRT cells were infected with RasV12 (300, 1,000, and 2,500 particles/cell) or LacZ (1,000 particles/cell) virus overnight, transferred into growth medium, and harvested after 48 h. Total cell lysates were analyzed for endogenous Rap1GAP and HA-Ras expression. Western blotting for AKT1 confirmed equal protein loading. Three experiments were performed, with similar results. (B) WRT cells were infected overnight with RasV12 or LacZ virus (300 particles/cell), transferred into growth medium in the presence and absence of 10 μM UO126, and harvested 48 h later. Rap1GAP expression and HA-Ras expression were analyzed. Western blotting for AKT1 confirmed protein loading. Three experiments were performed, with similar results. (C) WRT cells were infected with viruses expressing RasV12S35, activated MEK1 (C/A MEK), and β-galactosidase (LacZ) (2,500 particles/cell), and Rap1GAP expression was analyzed at 48 h. Expression of HA-Ras and MEK1 and ERK activity (ERK-p) are shown. Equal protein loading was confirmed by analyzing AKT1 expression. Three experiments were performed, with similar results. (D) WRT cells were infected with RasV12 and LacZ (1,000 particles/cell) viruses overnight and then transferred into growth medium in the absence (−) and presence (+) of 10 μM UO126 for 48 h. Cells were harvested, total RNA was isolated, and Rap1GAP message levels were analyzed as described in the legend of Fig. 1B. Five experiments revealed that RasV12 decreased Rap1GAP mRNA. Two experiments revealed little or no effect of UO126 on Rap1GAP message levels.