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. 2020 Nov 20;11(11):995. doi: 10.1038/s41419-020-03205-2

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© The Author(s) 2020

Open Access This 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 license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license 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 license, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 5 — a Identification of proteins that associate with ASB16-AS1. Biotinylated ASB16-AS1 or biotinylated antisense ASB16-AS1 was incubated with SW-13 cell extracts and RNA pull-down was performed to identify the proteins that associate with ASB16-AS1. The lanes were excised and analyzed by mass spectrometry. The arrow indicates the potential protein that specifically associates with ASB16-AS1. b HuR protein binds with ASB16-AS1. Biotinylated ASB16-AS1 or its antisense sequence was incubated with SW-13 cell extracts and RNA pull-down following western blot was performed to detect the association of HuR with ASB16-AS1. GAPDH served as negative control. c RIP assays to verify the association between ASB16-AS1 and HuR protein. HuR antibody was incubated with SW-13 cell extracts and qRT-PCR was performed to test the relative amount of ASB16-AS1 associates with HuR protein. IgG was served as negative control. *P < 0.05 versus IgG group. d HuR protein binds with fragment 2 of ASB16-AS1. Biotinylated full length ASB16-AS1 or truncated ASB16-AS1 fragment was incubated with cell extracts from SW-13 cells and RNA pull-down following western blot was performed using HuR antibody. Biotinylated antisense ASB16-AS1 was used as control. e, f Knockdown of ASB16-AS1 elevated HuR protein levels. ASB16-AS1 siRNAs were transfected into SW-13 cells, HuR mRNA levels was tested by qRT-PCR (e) and protein levels were tested by western blot (f). g, h Enhanced expression of ASB16-AS1 reduces the expression of HuR protein. ASB16-AS1 overexpression plasmid was transfected into SW-13 cells, HuR mRNA and protein levels were tested by qRT-PCR and western blot, respectively. pcDNA3.1 served as control. The experiments were performed in triplicate. The data are represented as mean ± SEM from three independent experiments.