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 Nov 15;8:16913. doi: 10.1038/s41598-018-35277-8

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

The nature of the third nucleotide influences protein lifetimes. (a–d) We designed four additional families of protein turnover sensors composed of the sequence of four different proteins fused to the SNAP-tag. For each one of the four protein species, we optimized five versions of their coding sequences with increasing percentages of G-/C-ending codons (0%, 25%, 50%, 75% and 100%), corresponding to 20 additional constructs. For each protein, the five plasmids with increasing percentages of G-/C-ending codons were separately transfected in COS7 cells in equimolar quantities. Also in this case, as for Fig. 4b, the sensors were pulsed for 2 h and chased for 24 h, revealing the turnover of the different proteins. For each desired chase time, cells were fixed, imaged with a high content microscope and the fluorescence analyzed in an automated fashion. The segmented lines represent the percentage of the dye that was lost due to the dilution caused by cell division from the beginning of the experiment. Each dot in the graphs represents the average of three separate experiments with SEM (n = 3). The inset in the upper right corner of each graph shows the relative protein expression at the beginning of the experiment (time₀), measured as absolute fluorescence of every sensor. Each panel shows the results from >10.000 cells analyzed. (e) Summary of the lifetimes (expressed as t_1/2), calculated through the fitting of the data represented in in Fig. 4b for Synuclein (Snca) and in panels a–d for the remaining proteins. The r² indicates the coefficient of determination for each fitting used for the calculation of the lifetimes. (f) Lifetimes increase versus the 0% GC3 condition. Higher G-/C-ending codon percentages continuously increase the t_1/2 from 0% to 100% GC3 (statistically significant trend with a linear regression P < 0.0001). With respect to the 0% GC3, in the 100% GC3 the lifetimes of these sensors are increased on average up to 229.0 ± 24%, roughly doubling their stability. ANOVA followed by Bonferroni post-hoc comparisons test: **P < 0.01, ***P < 0.001. Error bars = s.e.m.