Fig 3. Okadaic acid increases phosphorylation and cytosolic localization of FoxO1 in T regulatory cells.

(a) Primary CD4⁺ T cells were isolated by negative magnetic selection from the lymph nodes of FoxP3-GFP mice. Cells were cultured on plates coated with anti-CD3 and anti-CD28 and treated with TGFβ to initiate Treg skewing. Okadaic acid was added after 24 hours in culture. After 72 hours in culture, cells were removed from TCR stimulus for 2 hours prior to protein isolation. (b) Treg differentiation was assessed at the end of the assay by GFP expression. (c) Representative western blot of FoxO1 phosphorylation at Thr24 in Treg cells. Data are representative of 3 independent experiments. (d) Representative western blot of FoxO1 in fractionated Treg cells. Beta actin and HDAC were used as cytosolic and nuclear controls respectively. Bar graph shows summary localization data from 3 experiments as measured by band density and normalized to cytosolic control.