Figure 2

LSD1 is a bona fide demethylase for OCT4-K222. (A) Comparison of methylated lysine and arginine residues in non-treated recombinant OCT4 proteins with those in recombinant OCT4 proteins incubated with the lysates from different cell types. M, mono-methylation; Di, di-methylation; K, lysine; R, arginine. (B) Immunoblotting analysis of LSD1 protein levels in multiple human cell lines that include non-transformed differentiated cells, cancer cells, an ESC and an ECC. (C–E) Whole cell lysates of multiple cell lines were immunoblotted with anti-LSD1 and anti-OCT4 (C), and their LSD1 mRNA levels (D) and OCT4 mRNA levels (E) were determined by qRT-PCR. (F,G) H1 (F) and H9 (G) hESCs were induced to NSCs for 8 and 15 days, and the whole cell lysates were immunoblotted with the indicated antibodies. (H,I) Endogenous OCT4 proteins in NCCIT cells (H) and Myc-OCT4 proteins ectopically-expressed in 293 T cells (I) were immunoprecipitated with specific antibodies and immunoblotted with the indicated antibodies. (J) Co-localization of endogenous OCT4 and LSD1 proteins in NCCIT cells. White arrows marked the co-localization spots in different cells forming a single colony. (K,L) Recombinant His-OCT4 proteins were incubated with recombinant His-LSD1 proteins in the presence (K) or absence (L) of U87 whole cell lysates in PMA buffer for 1 h and subjected for MS analysis. The peak profiles of the peptides spanning the di-methylated and mono-methylated OCT4-K222 were presented. Cropped immunoblot images were presented for (B), (C), (F), (G), (H) and (I).