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. 2020 Jun 17;13:78. doi: 10.1186/s13045-020-00909-y

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© The Author(s) 2020

Open AccessThis article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data.

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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