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. 2018 Jun 25;9:2464. doi: 10.1038/s41467-018-04815-3

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© 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/.

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Fig. 6 — Smurf1 targets SRSF5 for degradation upon low glucose intake. a Smurf1 negatively regulates SRSF5 protein level in a dose-dependent manner. HEK293T cells were transfected with increasing amounts of Flag–Smurf1 WT and or Flag–Smurf1 CA vectors along with Myc-SRSF5 vectors for immunoblotting analysis. b Smurf1 knockdown increases the expression level of SRSF5 in A549 and H358 cells. c HEK293T cells transfected with the indicated plasmids were treated with CHX and harvested at the indicated times for western blot. d A549 cells transfected with siSmurf1 or control as previously described were treated with CHX for the indicated time to determine endogenous SRSF5 expression levels. e Expression analysis of endogenous SRSF5 protein in Smurf1^+/+, Smurf1^+/−, and Smurf1^−/− MEFs were revealed by immunoblotting. f Half-life analysis of SRSF5 in Smurf1^+/+ and Smurf1^−/− MEFs. g Expression levels of CCAR1 splice variants were examined in the lung tissues of Smurf1^+/+ and Smurf1^−/− mice by RT-PCR. h Co-immunoprecipitation assay revealed that endogenous SRSF5 interacts with Smurf1 in A549 cells. i GST pull-down assays were performed to indicate the direct interaction between Smurf1 and SRSF5. j The expression level of Smurf1 reversely correlates with SRSF5 when glucose concentration declines. k Glucose deprivation dampens endogenous Smurf1 ubiquitylation. A549 cells were maintained in medium with or without 25 mM glucose. The endogenous Smurf1 ubiquitylation level were determined by IP-western. l Knockdown of Smurf1 decreases SRSF5 ubiquitylation. HEK293T cells with or without siSmurf1 were transfected with indicated plasmids. The ubiquitylation of SRSF5 was determined by IB analysis. m Smurf1 promotes the K48-linked poly-ubiquitylation of SRSF5 in vivo. The SRSF5 ubiquitylation linkage was analyzed in HEK293T cells transfected with indicated plasmids. n Ectopic expression of hSmurf1 by lentiviral infection rescues the ubiquitylation of endogenous SRSF5 in Smurf1^−/− MEFs. o Smurf1 ubiquitylates SRSF5 by sensing glucose concentration. Smurf1^+/+ and Smurf1^−/− MEFs were maintained under various glucose concentrations and ubiquitylated SRSF5 was visualized. Blots are representative of three independent biological replicates. Error bars in c, d, f show s.e.m. from three independent experiments (**P < 0.01, two-way ANOVA test). Unprocessed original scans of blots are shown in Supplementary Fig. 9