Ub-enriched proteins induced a tumor-specific immune response. C57/BL16 mice (3 mice/group) were vaccinated respectively with 0, 30, 100 and 300 μg by Ub-enriched proteins isolated from EL4 whole cell lysate via triple s.c. injections (A) or i.n. priming injection plus twice s.c. injections (B) with two days interval. On day 10 after last vaccinations, lymphocytes were collected from spleen and lymph nodes and re-stimulated by EL4 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) Comparing between the immune response resulted by previous two different route of vaccination (s.c. and i.n.) respectively using 100 μg of Ub-enriched proteins. Data are representative of three independent experiments results.