Fig. 5.

Depletion of Tregs abolishes WP1066-enhanced phosphorylation of ZAP-70 (p-ZAP-70) in CD3+ T cells. CD3+ and CD3+CD25− T cells were isolated from the PBMCs of healthy donors, and melanoma brain metastasis patients. The cells were incubated with either the RPMI 1640 medium alone or supplemented with WP1066. Subsequently, cells were lysed, electrophoretically fractionated in 8% SDS-polyacrylamide gels, transferred to nitrocellulose membranes, and immunoblotted with antibodies to p-ZAP-70 (Tyr319), ZAP-70, phosphorylated AKT (p-AKT: Ser473), AKT, or β-actin. These experiments were reproduced four times with similar results