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. 2018 Jun 6;50(6):1–10. doi: 10.1038/s12276-018-0102-5

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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. 3 — a Immunoblot analysis of SW48, D-WT, D-MT, HCT-116, and LoVo cells treated with 25 μM of KY7749 for 24 h. b Immunoblot analysis of HCT-116 cells treated with various doses of KY7749 for 24 h. c Elk-1 reporter assay in HCT-116 cells treated with 25 μM KY7749 for 24 h. The effect of KY7749 on ERK activity was detected based on the activity of an Elk-1 reporter construct. d SW480 cells were treated with 25 μM KY7749 for 24 h. GTP-bound Ras was measured as described in the Materials and Methods by pull-down of GTP-bound Ras and subsequent monitoring of phosphorylation of the Raf-1 Ras-binding domain (GST-RBD). e Immunoblot analysis of SW480 CRC cells overexpressing the MEK1CA construct and treated with 25 μM KY7749 for 24 h. f MTT assays in MEK1 CA-overexpressing SW480 CRC cells cultured with DMSO (control) or various doses of KY7749 for 96 h (mean ± SD, n = 3). Relative cell proliferation was normalized to DMSO-treated controls. WCLs or pull-down samples were immunoblotted with antibodies against the indicated proteins (a–b, d–e)