Fig. 3
Identification and characterization of LAMP5-AS1 binding to DOT1L. a Western blotting and silver staining of SDS-PAGE followed by mass spectrometry showed that DOT1L was enriched by LAMP5-AS1 pull-down assays. Beta-tubulin as the negative control. b LAMP5-AS1 was significantly enriched by the RIP of DOT1L in MLL leukemia cells. Error bars reflect ± SEM from three independent experiments. c RNA FISH and IF experiments showed that LAMP5-AS1 co-localized with DOT1L in the cell nucleus. Scale bar, 5 μm. d RIP-qPCR for the LAMP5-AS1 and FLAG-tagged DOT1L sections. DOT1L (1-416 aa, 1-822 aa, and 1-1537 aa) presented significantly higher enrichment of LAMP5-AS1. Error bars reflect ± SEM from three independent experiments. Immunoblotting indicated the successful RIP assay. e, f Immunoblot detection of HA-tagged DOT1L (1-416 aa) retrieved by in vitro-transcribed tRSA-tagged LAMP5-AS1 sections from 293T cell lysates. LAMP5-AS1 (503-1036 nt, 1-1036 nt, and 1-1928 nt) presented significant enrichment. Beta-tubulin or lamina A/C as the negative control. g RIP-qPCR detection for the enrichment of FLAG-tagged DOT1L (1-416 aa) on LAMP5-AS1 truncated mutants in 293T cells. LAMP5-AS1 (503-1036 nt) presented significantly higher enrichment. Immunoblotting indicated the successful RIP assay. Error bars reflect ± SEM from three independent experiments. h Immunoblot detection of HA-tagged DOT1L (1-416 aa) sections retrieved by in vitro-transcribed tRSA-tagged LAMP5-AS1 (503-1036 nt) from 293T cell lysates. DOT1L (1-407 aa, 360-416 aa, and 1-416 aa) presented significantly higher enrichment of LAMP5-AS1 (503-1036 nt). Lamina A/C as the negative control. i RIP-qPCR detection for the enrichment of HA-tagged DOT1L (1-416 aa) sections on LAMP5-AS1 (503-1036 nt) in 293T cells. LAMP5-AS1 (503-1036 nt) was highly enriched in DOT1L (1-407 aa, 360-416 aa, and 1-416 aa)-transfected 293T cell lysates. Error bars reflect ± SEM from three independent experiments. Immunoblotting indicated the successful RIP assay. Lamina A/C as the control