Figure - PMC

Skip to main content

An official website of the United States government

Here's how you know

Here's how you know

Official websites use .gov
A .gov website belongs to an official government organization in the United States.

Secure .gov websites use HTTPS
A lock ( ) or https:// means you've safely connected to the .gov website. Share sensitive information only on official, secure websites.

View full-text article in PMC

. 2018 Mar 26;9:1235. doi: 10.1038/s41467-018-03681-3

Search in PMC
Search in PubMed
View in NLM Catalog
Add to search

© The Author(s) 2018

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/.

PMC Copyright notice

Fig. 3 — ARPP21 acts as a positive regulator of post-transcriptional gene expression in a tethering assay and functionally interacts with parts of the eIF4F complex. a The MS2 coat protein was used to tether ARPP21 to the 3′ UTR of a GFP reporter transcript in 293T cells. b ARPP21 tethering results in increased GFP fluorescence of the MS2-binding site reporter (4X-MS2-BS-GFP) compared to tethering of the MS2 protein alone. Tethering of the translational inhibitor AGO2 decreases basal GFP fluorescence. Scale bar: 50 µm. c Flow cytometry analysis of tethering assays, expressed as mean fluorescent intensity (MFI) of transfected cells. AGO2-MS2 decreases, and ARPP21-MS2 and R3HDM1-MS2 increase the expression of the 4X-MS2-BS-GFP reporter, relative to Flag-MS2 alone. The C-terminal part of ARPP21 is necessary and sufficient for the positive effect on reporter expression. ***p < 0.001, Student’s t-test, n = 3, error bars represent ± s.d. d Untethered ARPP21 and R3HDM1 have no significant effect on GFP expression from the 4X-MS2-BS-GFP reporter relative to MS2 control. Statistical analysis as in c, n = 3. e FLAG-tagged ARPP21 or R3HDM1 were immunoprecipitated from 293T lysates after RNase digestion and protein interactors were identified by LC-MS/MS. f Co-immunoprecipitation (co-IP) using FLAG- or GFP-tagged ARPP21 constructs verify the ability of ARPP21 to self-interact by reciprocal co-IP. RNAse treatment strongly reduces co-IP of the RNA-binding protein IMP1 but not homomeric ARPP21 interactions. Loss of GAPDH protein after IP is shown as control. g FLAG-ARPP21 coimmunoprecipitates endogenous eIF4A and eIF4G from N2A cells. Antibodies used for detection of proteins in input and eluate are shown on the left. RNase pretreatment of lysates had no effect on co-IP of eIF4A or eIF4G. h, i ARPP21 and R3HDM1 coimmunoprecipitate eIF4A1 more efficiently than eIF4A2. HA-tagged ARPP21 or R3HDM1 was co-transfected with FLAG-tagged eIF4A1 (h) or eIF4A2 (i); results after immunoprecipitation with anti-HA antibody are shown. j Validation of endogenous eIF4G1 knockdown in 293T cells used in tethering assay. k ARPP21 activity in the tethering assay is reduced after eIF4G knockdown. ***p < 0.001, two-way ANOVA with Bonferroni post-test, n = 4, error bars represent s.d. Full western blot images are presented in Supplementary Fig. 21