Figure 2.

H₂O₂ induces Ral activation and Ral activation is necessary and sufficient for H₂O₂-induced T451 phosphorylation. (A) A14 cells were treated with 100 μM of H₂O₂ for the indicated time, and Ral GTP levels were analyzed on Western blot using a Ral pull-down assay (upper panel). The lower panel shows endogenous Ral protein levels. Same results were obtained using 200 and 400 μM H₂O₂. (B) 293T cells, transfected with HA-FOXO4 and HA-RalN28 or a control construct, were treated with 100 μM H₂O₂ for the indicated time and T451 phosphorylation was analyzed on Western blot. Same results were obtained using 200 or 400 μM H₂O₂. (C) 293T cells were transfected with myc-FOXO4 together with the indicated constructs. T451 phosphorylation was analyzed.