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 Oct 8;9:4139. doi: 10.1038/s41467-018-06556-9

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. 1 — RhoB is sumoylated by PIAS1. a RhoB interacts with Ubc9. HEK293T cells transfected with indicated combinations of Flag-tagged Ubc9 (F-Ubc9) and HA-tagged RhoB (HA-RhoB) were subjected to anti-Flag immunoprecipitation (IP) followed by immunoblotting assay to detect associated RhoB. b RhoB is sumoylated. HEK293T transfected with indicated combinations of His-tagged RhoB (His-RhoB), HA-tagged SUMO (HA-SUMO) 1 or 2, and Myc-tagged Ubc9 (Myc-Ubc9) were lysed with 6 M guanidine-HCl followed by Ni-NTA agarose beads pull-down (Ni pull-down) assay. SUMO-conjugated RhoB was detected by immunoblotting with anti-HA. Conjugation of mono-SUMO and multi-SUMO to RhoB are indicated as RhoB-SUMO and RhoB-(SUMO)_n, respectively. c PIAS1 promotes sumoylation of RhoB. HEK293T cells cotransfected with His-RhoB, HA-SUMO2, and indicated Myc-tagged PIAS (Myc-PIAS) family member 1–4 were subjected to sumoylation assay as described in panel b. d Knockdown of PIAS1 attenuates RhoB sumoylation. HEK293T cells transfected with indicated combinations of His-RhoB, HA-SUMO2, and shRNAs against PIAS1 were subjected to sumoylation assay as described in panel b. e The E3 catalytic activity is required for PIAS1-mediated RhoB sumoylation. HEK293T cells were transfected with His-RhoB, HA-SUMO2, and Myc-PIAS1 wild-type (WT) or catalytically inactive mutant (C351S) as indicated. The cells were subjected to sumoylation assay as described in panel b. f PIAS1 interacts with endogenous RhoB. HeLa cells transduced with lentivirus encoding HA-tagged PIAS1-C351S mutant (HA-PIAS1-C351S) were subjected to anti-RhoB IP followed by immunoblotting with rat anti-HA to detect associated HA-PIAS1-C351S. g Sumoylation sites mapping on RhoB. HEK293T cells transfected with HA-SUMO2 and indicated His-RhoB mutants were subjected to sumoylation assay as described in panel b. h PIAS1 enhances sumoylation of WT but not 4KR RhoB. HEK293T cells transfected with indicated combinations of HA-SUMO2, Myc-PIAS1 (WT or C351S), and His-RhoB (WT or 4KR) were subjected to sumoylation assay as described in panel b