Lyn Tyr⁵⁰⁷ dephosphorylation and Tyr³⁹⁶ phosphorylation occurred simultaneously with the rapid translocation of CD45RO to lipid rafts. (A) U266 CD45^-, U266 CD45⁺, NOP2, ILKM2, U266 EGFP, and U266 CD45RO-, CD45RB-, and CD45RA-EGFP cells stimulated with or without IL-6 (10 ng/mL) for 5 minutes were collected and whole-cell lysates were subjected to immunoblotting with antibodies against CD45, P-Lyn Tyr⁵⁰⁷, and the P-Src family (Tyr⁴¹⁶). Control blots were performed with antibodies against Lyn. (B) U266 CD45⁺ cells starved of IL-6 for 12 hours were stimulated with 10 ng/mL IL-6 for 0.5 and 5 minutes, and then samples of the raft fraction from each point were immunoblotted with antibodies against CD45RO, CD45RB, P-Lyn Tyr⁵⁰⁷, the P-Src family, and Lyn. (C) U266 CD45⁺ cells cultured with IL-6 were starved of IL-6 by replacing the medium using centrifugation, and the cells refed with culture medium were recovered at 0.5 and 5 minutes. Samples of the raft fraction from each point were immunoblotted using the same antibodies as in panel B.