Figure 2. Lys 322 on LSD1 methylation by SUV39H2 both in vitro and in vivo.

A. In vitro methyltransferase assay indicated that LSD1 peptide (amino acid residues 313-330) was methylated by SUV39H2 but not Lys 322-substituted LSD1 peptide (K322R). Amounts of loading proteins were evaluated by staining the MemCode^TM Reversible Protein Stain (Thermo Fisher Scientific). B. Amino acid sequence indicated that the methylation site Lys 322 was highly conserved across species. C. Methylation of LSD1 in human cells was confirmed by in vivo labeling experiment. 293T cells were transfected with FLAG-LSD1-WT or FLAG-LSD1-K322R in the presence of HA-SUV39H2 and treated with methionine-free medium, including cycloheximide and chloramphenicol. They were then labeled with L-[methyl-³H] methionine for 3 hours. Cell lysates were immunoprecipitated with FLAG-M2 agarose, and methylated LSD1 was visualized by fluorography. The membrane was immunoblotted with an anti-FLAG (an internal control) antibody.