CD33 and SOCS3 interaction. (A) 293T cells were transfected with 2 μg SOCS3 pME18S-Flag. (Ai) Lysates were coprecipitated with appropriate CD33 peptide and immunoblotted with α-Flag (M2), or (Aii) the lysates were immunoprecipitated with α-Flag and immunoblotted with α-Flag. (Ai-ii) Peptide sequences in lanes 1 to 8 were as follows: (1,3) phosphorylated ITIM peptide (biotin SGSGDEELHpYASLNF-OH); (2,4) unphosphorylated ITIM peptide (biotin SGSGDEELHYASLNF-OH); (5) phosphorylated ITSM peptide (biotin SGSGDTSTEpYSEVRT-OH); (6) unphosphorylated ITSM peptide (biotin SGSGDTSTEYSEVRT-OH); (7) control peptide (biotin SGSGGHDGLYQGLST-OH); and (8) positive SOCS3 control (IP α-Flag). (B) Signal intensity of SOCS3 levels pulled down by pY ITIM and pY ITSM peptides were normalized to SOCS3 expression levels and are illustrated as a graph.