Fig. 4

Effect of LAMP5-AS1 on the HMTase activity of DOT1L. a GST-tagged DOT1L (1-416 aa) overexpressed in one-shot BL21(DE3)-competent E. coli cells and purified using GST affinity purification and then digested with thrombin is shown by Coomassie staining. GST-tagged DOT1L (1-416 aa), DOT1L (1-416 aa), and GST are presented. b Electrophoresis of RNA on gel containing formaldehyde, showing LAMP5-AS1 results from RNA transcription in vitro. c Schematic depiction of the experimental design. d Representative HMT assay checked by H3K79me2 and H3K79me3 immunoblotting showed that LAMP5-AS1 enhanced the HMTase activity of DOT1L. Purified HeLa nucleosome was used in the assay. e Binding of the active domain (503-764 nt) of LAMP5-AS1 to DOT1L (1-416 aa) measured by electrophoretic mobility shift assay (EMSA)