K132 is important for OCT4 chromatin association after CoCl₂ treatment.

<p>A. HEK293 cells were transfected with WT OCT4 or OCT4^K123R for 48 h and then treated with CoCl₂ for various times. Equal amounts of cell lysates were then blotted for OCT4 and α-tubulin. B. HEK293 cells were transfected with WT OCT4 or various mutant constructs for 48 h followed by treatment with CoCl₂ for 3 h. Cells with various treatments were lysed and separated into cytoplasmic (Cy), soluble nuclear (N) and chromatin (Ch) fractions. Equal amounts of proteins from each fraction were blotted with antibodies to OCT4 and α-tubulin. C. HEK293 cells transfected with WT OCT4 for 48 h were treated with CoCl₂ (0, 50, 100, 150, and 200 µM) and/or ascorbic acid (AA) (0, 50, 100, 150, and 200 µM) for 3 h. At the end of treatments, cells were lysed and equal amounts of proteins were blotted for OCT4 and α-tubulin. D. HEK293 cells were transfected with WT OCT4 or various mutant constructs for 48 h followed by treatment with CoCl₂ or ascorbic acid (AA) for 3 h. GFP expression plasmid was used for co-transfection to monitor transfection efficiency. Cell lysates of various treatments were blotted with antibodies to OCT4, GFP and α-tubulin. Both short and long exposures of OCT4 blots were shown. S-OCT4 and U-OCT4 denote sumoylated and ubiquitinated OCT4, respectively.