Figure 5: p53 interacts with C-terminal domain of 14-3-3γ. Human 14-3-3γ fragments (1: 1-78 amino acid, N-terminal; 2: 79-131 amino acid, central part; 3: 132-184 amino acid, central part; and 4: 185-247 amino acid, C-terminal) were amplified by PCR and inserted in-frame into pGEX2T vector to make truncated constructs. The GST-fusion proteins were expressed in bacterial and purified with glutathione sepharose 4B beads. Equal amount of fusion proteins were loaded on SDS-PAGE and then stained with Coomassie blue (A). The purified GST and GST-14-3-3γ fragments were incubated with 200 µg of HCT116 cell lysate for 4 h at 4°C. The complex was pulled down with 40 µl of glutathione sepharose beads for 2 h at 4°C. After five washes with lysis buffer, the bound proteins were subjected to SDS-PAGE, transferred to nitrocellulose membrane, and immunoblotted with anti-p53 antibody (B).