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. 2016 Aug 22;7:12417. doi: 10.1038/ncomms12417

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Copyright © 2016, The Author(s)

This work is licensed under a Creative Commons Attribution 4.0 International License. The images or other third party material in this article are included in the article's Creative Commons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission from the license holder to reproduce the material. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/

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(a) RhoA activity was assessed in hPASMCs transduced with LacZ, Cav1-HA and Cav1-HA and MURC-FLAG (n=4 per group). hPASMCs were infected with a hygromycin-resistant retrovirus expressing LacZ and Cav1-HA. After selection using hygromycin, hPASMCs were infected with a puromycin-resistant retrovirus expressing LacZ and MURC-FLAG, and subsequently selected using puromycin. *P<0.05 compared with LacZ+LacZ, ^†P<0.05 compared with LacZ+Cav1. (b) COS cells were transfected with pcDNA3.1-hMURC-HA and/or pcDNA3.1-hGα13. The COS cell lysates incubated with GDP or GTPγS and GST pulldown was performed with GST-fusion Cav1 conjugated to glutathione-Sepharose beads. Precipitated proteins were blotted with anti-Gα13 and anti-GST antibodies (n=3 per group). *P<0.05 compared with Gα13-GDP+Cav1, ^†P<0.05 compared with Gα13-GTPγS+Cav1. (c) The association of Gα13 with Cav1 was assessed in hPASMCs transfected with control siRNA or MURC siRNA (n=3 per group). *P<0.05 compared with control siRNA. (d) GST-fusion Gα13 conjugated to glutathione-Sepharose beads was preloaded with GDP or GTPγS. GST pulldown was performed with COS cell lysates transfected with plasmids expressing the indicated proteins. Precipitated proteins were blotted with anti-FLAG and anti-GST antibodies (n=5 per group). *P<0.05 compared with Gα13-GDP+p115RhoGEF, ^†P<0.05 compared with Gα13-GTPγS+p115RhoGEF, ^#P<0.05 compared with Gα13-GTPγS+p115RhoGEF+Cav1. (e) Each domain of Cav1 (FL: full length, 1–61: C-terminal domain, 61–101: oligomerization domain, 135–178: N-terminal domain) tagged with GST was conjugated to glutathione-Sepharose beads, and incubated with lysates from COS cells transfected with pcDNA3.1-hMURC-FLAG or pcDNA3.1-hGα13. Pulled-down protein was blotted with anti-MURC or anti-Gα13 antibodies. Arrows indicate each GST-fusion protein. Data are presented as mean±s.e.m. Uncropped images of blots are shown in Supplementary Fig. 6.