TCTP phosphorylation by insulin does not occur at Ser-46, -64, and -98 residues. Following transfection of 293T cells with pEGFP-N1-TCTP (WT) or pEGFP-N1-TCTPS46AS64AS98A (triple mutant, TM), cells were incubated with insulin-containing media. In order to determine whether Ser-46, -64, and -98 residues are involved in the phosphorylation sites for TCTP, alteration of p-Ser-TCTP in cytosolic fractions was assessed and compared in the presence or absence of Ser to Ala mutation in TCTP sequence. Following immunoprecipitation with anti-p-Ser antibody, immune complex was resolved and immunoblotted using anti-GFP antibody.