Figure 1
K222 site of human OCT4 protein can be methylated in vitro and in vivo. (A) Schematic representation of the cell-free system-based in vitro PTM analysis approach. (B) 1 μg of purified E. coli-derived His-OCT4 protein binding to the Ni–NTA beads (20 μl) was incubated with 0.2 mg of NCCIT whole cell lysate in 1 ml PMA buffer at 30℃ for varying periods from 0 to 180 min. Samples were subjected to SDS-PAGE and immunoblotted with the indicated antibodies. (C) Summary of the PTM profiles of recombinant His-OCT4 proteins in the cellular contexts of NCCIT and U87 cells. (D,E) NCCIT whole cell lysates were immunoprecipitated with anti-OCT4, and immunoblotted with anti-pan K(me/me2) (D) or anti-OCT4-pT235 (E). Cropped immunoblot images were presented. (F) Schematic representation of the OCT4 protein structure, highlighting the methylated K222 site at the linker region that connects the POUS with POUH domains. Multiple alignment of amino acid sequences at the linker region indicated that the K222 is a highly conserved residue across multiple species.
