Figure 3. Rac1GTPase and Cdc42GTPase are involved in the early cofilin phosphorylation.

A. ROCK inhibitor Y27632 has no effect on the entry of TGEV. IPEC-J2 cells were pretreated with Y27032 at different concentrations at 37°C for 1 h. TGEV binding and entry assays are described in materials and methods. B. PAKs inhibitor IPA-3 does inhibit TGEV entry. IPEC-J2 cells were pretreated with IPA-3 at different concentrations at 37°C for 1 h. C. PAKs are involved in the phosphorylation of cofilin. Cells were pretreated with Rock inhibitor Y27632 (100μM) and PAKs inhibitor IPA-3 (50μM) at 37°C for 1 h. 30 min after TGEV infection, cell lysates were analyzed for phosphorylation of LIMK and Cofilin. (D to F) RHO-family-GTPases are involved in the entry of TGEV. Cells were treated with lentiviral particles expressing WT, constitutively-activated, and constitutively-inactivated RHO GTPases. After 48 h incubation, cells were infected with TGEV for 1 h. Entry of TGEV was evaluated by real-time PCR. (G to I) Rac1 and Cdc42 are involved in the regulation of cofilin. Cells were treated with lentiviral particles expressing WT, constitutively-activated, and constitutively-inactivated RHO GTPases. After 48 h incubation, cells were infected with TGEV for 30 min and cell lysates were analyzed for the phosphorylation of LIMK and Cofilin. J. IPA-3 protects the actin cytoskeleton. Cells were treated with Y27632 (100 μM), IPA-3 (50 μM) at 37°C for 1 h, then infected with TGEV. 1 h after TGEV infection, cells were fixed and stained with phalloidin-TRITC and observed by confocal fluorescence microscopy. TGEV at a multiplicity of infection of 2 (MOI = 2).