Figure 3

Rotavirus VP8* protein triggers activation of the RhoA/ROCK/MLC pathway. (a and b) Confluent MDCK monolayers were incubated with recombinant GST-tagged VP8* protein of RVA strain (a) DS-1 or (b) NCDV (10 μg/ml) for the indicated time points. Cell lysates were 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 check the expression level of ROCK, pMLC, and MLC2 using the relevant antibodies. GAPDH was used as a loading control. (c and d) Confluent MDCK monolayers were either mock-treated or pretreated with (c) RhoA inhibitor CT04 or (d) ROCK inhibitor Y27632 for 1 h at 37 °C and then incubated with the purified VP8* protein of RVA strain DS-1 or NCDV (10 μg/ml). Cell lysates were harvested at 1 h and the expression levels of ROCK and/or pMLC were evaluated by western blot analysis using the relevant antibodies. GAPDH was used as a loading control. All experiments were performed in triplicate and representative images of different gels from each group are presented. The intensities of RhoA, ROCK, and pMLC relative to GAPDH were determined by densitometric analysis and are indicated above each lane.