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. 2020 Apr 3;11:1674. doi: 10.1038/s41467-020-15412-8

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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. 4 — a Relative mRNA levels determined by qRT-PCR of pan-Elavl4 after either Control (Ctrl), Celf1L, or Celf1S overexpression (OE) in N2a cells (n = 6 transfections). Data represent the mean and SEM. Data normalized to Gapdh and then to Ctrl. Statistics: one-way ANOVA. n.s. = not significant. b (left) Western blot for Ctrl, Celf1L, or Celf1S OE in N2a cells (n = 6 transfections). Immunoblot of pan-Elavl4 (upper panel). Gapdh is the loading control (lower panel). (right) Densitometry quantification of Western blots. Data represent the mean, SD. Data normalized to Gapdh and then to Ctrl. Statistics: one-way ANOVA. *p < 0.05. c Schematic of 3′ UTR luciferase construct (left of bar graph). Elavl4 isoforms’ common 3′ UTR downstream of Luciferase (Luc), and cotransfected with either Ctrl OE/Celf1S into N2a cells. Translation assayed by measuring LUC RLU over Luc mRNA levels (n = 9 transfections). Data represent the mean and SEM. Statistics: Student’s t-test. n.s. = not significant. d Schematic of 5′ UTR Renilla construct (left of bar graph). N2a cells were cotransfected with Ctrl OE or Celf1S and Elavl4-v2 5′ UTR upstream of Renilla (Ren). Same assay as in c; REN measured over Ren mRNA (n = 3 transfections). Data represent the mean and SEM. Statistics: Student’s t-test. n.s. = not significant. e, f Deletion strategy of Celf1-regulatory regions in 5′ UTRs of Elavl4-v3 (e) and Elavl4-v1&4 (f). Elavl4-v3 or Elavl4-v1&4 5′ UTRs Renilla constructs; same strategy as in d. Dashed lines represent deleted regions. Numbers indicate positions in UTR, 5′ → 3′. n = 4 transfections for each experiment. bp = base pairs. Putative Celf1 regulatory sequence in Elavl4 5′ UTRs indicated under their respective bar graphs. Data represent the mean and SEM. Statistics: Student’s t-test and one-way ANOVA with Tukey’s (e, f). *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001; n.s. = not significant. g, h Celf1 RIP of Elavl4-v3 (g) and Elavl4-v1&4 (h) 5′ UTRs. UTRs have WT, mutated (Mut), or deleted (Del) Celf1 binding sequences. 18s was used as negative control. Data represent the mean and SEM; normalized to Gapdh. n = 3 RIPs from 3 transfections; Statistics: Two-way ANOVA with Tukey’s. *p < 0.05, **p < 0.01, ***p < 0.001.