Tumor specific immune response resulted by Whole tumor cell lysate, Ub-depleted proteins and Ub-enriched proteins. C57/BL6 mice (3 mice/group) were primed by 30 μg i.n and twice 100 μg s.c vaccination with two days of interval by whole cell lysate, Ub-depleted proteins and Ub-enriched proteins from EL4 tumor cells (A, C) and B16-F10 tumor cells (B, D). On day 10 after last vaccinations, blood was collected and the IFN-γ levels in the sera were detected by ELISA (A, B). Lymphocytes were collected from spleen and lymph nodes and re-stimulated by EL4 and B16-F10 tumor cells that already inactivated by 10 nM of colchicine or without stimulation (CM). α-CD3-Ab stimulation was used as positive control. The IFN-γ produced by the responder cells was determined after 72 h by ELISA (C, D). Data are representative of three independent experiments results. (E) Flow cytometric analysis of intracellular IFN-γ synthesized by CD4+ T cells (upper panel) and CD8+ T cells (lower panel) in the lymphocytes from mice vaccinated with PBS and whole cell lysate, Ub-depleted proteins and Ub-enriched proteins from B16-F10 tumor cells and restimulated with inactivated B16-F10tumor cells. Numbers within quadrants represent the percentage of positive cells for IFN-γ within the gate for relevant lymphocytes.