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. 2018 Nov 16;8:16932. doi: 10.1038/s41598-018-35237-2

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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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FLN-induced N-Ras degradation is mainly proteasome-independent. (A) MDCK cells stably expressing a fluorescently tagged N-Ras protein (GFP-N-Ras, green) were examined by microscopy after FLN treatment, and DAPI (blue) was added to mark the nucleus. (B) Western blot analysis of SUM149PT cells treated by FLN (20 µM, 48 hours). H-Ras, K-Ras, Cdc42, and GAPDH were examined as controls. Original full length gels for this and other figures can be found in Supplemental Information. (C) SUM149PT cells were pre-treated with vehicle or FLN, and time points were taken and analyzed by Western blot after CHX addition (Left). A representative immunoblot 10 hours post-CHX treatment is shown on the right. Numbers below are GAPDH-normalized N-Ras levels relative to that in the vehicle control. (D) N-RAS mRNA time points in panel-C were also analyzed by semi-quantitative RT-PCR, normalized by levels of control 36B4 mRNA. (E) Left, SUM149PT cells treated with indicated drugs for 6 hours were analyzed by Western blot. The numbers show N-Ras levels relative to those in cells not treated by FLN. Data from 3 separate experiments are quantified on the right.