Translocated CD45 complexed with IL-6Rα and Lyn, and PAG in lipid rafts. (A) U266 CD45⁺ cells starved of IL-6 for 12 hours were treated (+) or not treated (-) with IL-6 (10 ng/mL) for 5 minutes and separated by sucrose fractionation. The fractions of lipid rafts or nonrafts were mixed and immunoprecipitated with anti-CD45 antibody after adding 1% octyl-glucoside. Precipitated protein was immunoblotted with antibodies against IL-6Rα, Lyn, flotillin-2, or CD45. Control blots were performed using the raft or nonraft mixture directly with the antibodies indicated. (B) Fractions of cell lysates prepared as in panel A were subjected to immunoblotting with antibodies against Csk, PAG, and CD45. Then, 1% octyl-glucoside-added lipid raft fractions were immunoprecipitated with anti-PAG antibody. Precipitated proteins were immunoblotted with antibodies against phosphotyrosine 4G10 (detects phosphorylated PAG according to the size of the band), Csk, CD45, and PAG. IP indicates immunoprecipitation; WB, Western blot.