Figure 4. Cdk5 is required to induce Stat3 binding to the Foxp3 gene.

A) Protein lysates were isolated from the cytoplasmic and nuclear compartments of activated T cells with or without TGF-β and CIP treatment for 48hrs. Western blot analyses were performed probing for p-Stat3 (S727), p-Stat3 (Y705), total Stat3 and Cdk5. Both β-actin and YY-1 were probed as cytoplasmic and nuclear loading controls, respectively. B) Nuclear protein lysates were isolated from primary T cells activated by anti-CD3/CD28, with or without TGF-β, IL-6 and CIP treatment for 72hrs. Lysates were incubated with DNA probes that corresponded to the specific Stat3 binding region on the enhancer region II of the Foxp3 gene and allowed to complex in a DNA pull down assay. Western blot analyses were then performed for Stat3 bound to the DNA probes. C) Nuclear protein lysates were prepared from EL4 T cells stimulated under similar conditions, and from EL4 T cells expressing the Stat3 (S727A) mutant protein. Lysates were subjected to a DNA pull-down using DNA probes corresponding to the Stat3 binding enhancer II region of the Foxp3 gene. D) ChIP analysis was performed with lysates from primary T cells that were similarly stimulated with or without anti-CD3/CD28, with or without TGF-β, in the presence or absence of IL-6 and CIP. PCR was performed to amplify the specific crosslinked DNA corresponding to the Stat3 region on the Foxp3 enhancer II region. E) Similarly, ChIP analyses were performed with lysates from both Cdk5^+/+ T cells and Cdk5^−/− T cells activated in the presence or absence of either TGF-β and/or IL-6.