Figure 6

Distribution of tight junction proteins is altered by the rotavirus-induced RhoA/ROCK/MLC signaling pathway. (a–d) MDCK monolayers were untreated or treated with RhoA inhibitor (CT04), ROCK inhibitor (Y27632), or MLC inhibitor (blebbistatin) for 1 h at 37 °C and then mock-inoculated or inoculated with RVA strain (a and b) DS-1 or (c and d) NCDV (MOI = 10) for 1 h. Cells were then fixed, permeabilized, and prepared for confocal microscopy using rabbit anti-ZO-1, occludin, claudin, and JAM-A antibodies and relevant secondary antibodies. All experiments were performed in triplicate and representative images are shown. The scale bars correspond to 20 μm. (b and d) Quantification of the internalization of each TJ molecule (shown as a percentage of intracellular fluorescence/total fluorescence) as described in Materials and Methods.