Figure 4. The PI3K-Akt pathway is involved in the regulation of actin cytoskeleton by cofilin.

A. Activation of Akt by TGEV entry. Cells lysates were immunoblotted with anti-p-Akt and anti-Akt mAbs. B. Activation of ERK1/2 during TGEV entry. Cells lysates were immunoblotted with anti-p-ERK1/2 and anti-ERK1/2 mAbs. C. PI3K inhibitor LY294002 inhibits TGEV entry. IPEC-J2 cells were pretreated with LY294002 (50 μM) at 37°C for 1 h. Binding and entry assays are described in materials and methods. D. MEK1/2 inhibitor U0126 inhibits TGEV entry. IPEC-J2 cells were pretreated with U0126 at different concentrations at 37°C for 1 h. E. LY294002 and U0126 inhibit the activation of downstream pathways. Cells were treated with LY294002 (50 μM) or U0126 (250 μM) at 37°C for 1 h, then infected by TGEV. 30 minutes after infection, cell lysates were analyzed for the phosphorylation of Akt, ERK1/2, LIMK, and Cofilin. F. LY294002 and U0126 protect the actin cytoskeleton. Cells were treated with LY294002 (50 μM) or U0126 (250 μM) at 37°C for 1 h, then infected by TGEV. After 1 h, cells were fixed and stained with phalloidin-TRITC and observed by confocal fluorescence microscopy. Statistical significance was assessed by Student's t-test (*). Differences were considered to be significant at 0.01 < p < 0.05, (**) p < 0.01. All experiments were performed independently three times. Scale bar = 20 μm. TGEV at a multiplicity of infection of 2 (MOI = 2).