Fig 3.

The MEK inhibitor U0126 blocks the activation of ERK in RRV infection, inhibiting viral protein expression and virus production. (A) RhF were stimulated with TPA after 48 h of culture in the presence of drug vehicle DMSO (lane 1) or the MEK inhibitor U0126 (lane 2). RhF were cultured without exposure to RRV (lane 3; overloaded) or with exposure to RRV at an MOI of 5 in the presence of vehicle (lane 4) or U0126 (lane 5). For drug-treated samples, fresh inhibitor was added 24 h p.i. All cells were collected at 48 h p.i. for immunoblot analysis. Blots were probed with the following antibodies as indicated in the figure: anti-MCP, anti-ORF45, anti-pERK, anti-total ERK, anti-SCIP, and, to control for sample loading, anti-Ran. (B) Representative phase-contrast images of RhF that were uninfected (panel i) or infected with RRV (MOI of 5) and either pretreated with vehicle (panel ii) or U0126 (panel iii). (Original magnification for all panels, ×10). (C) Viral titers from the media collected under each condition as described for panel B. The columns represent the means ± SEM of the results of three independent experiments. UI, uninfected. (D) Values for viral genome equivalents in the media collected under each condition indicated were determined by qPCR. Data are from a representative experiment, with bars representing the SEM of the results from qPCR experiments performed in quadruplicate. ***, P < 0.001 (Student's t test).