Figure 2

The RhoA/ROCK signaling pathway is involved in rotavirus-induced early activation of pMLC. (a and b) Confluent MDCK monolayers were mock-infected or infected with RVA strain (a) DS-1 or (b) NCDV (MOI = 10) for the indicated time points. The cells were then harvested and subjected to a Rhotekin pull-down assay to measure RhoA activation and then analyzed by western blot analysis using an anti-RhoA antibody or directly analyzed by western blot analysis to examine the expression levels of ROCK using specific primary antibody. (c and d) Confluent MDCK monolayers were pretreated with or without (c) RhoA inhibitor (CT04) or (d) ROCK inhibitor (Y27632) at the indicated doses for 1 h at 37 °C and then infected with strain DS-1 or NCDV (MOI = 10). Cell lysates were harvested at 1 h post-infection, and the expression levels of ROCK and/or pMLC were evaluated by western blot analysis. GAPDH was used as a loading control. The intensities of RhoA, ROCK, and pMLC relative to GAPDH were determined by densitometric analysis and are indicated above each lane. All experiments were performed in triplicate and representative images of different gels from each group are presented.