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. 2018 May 16;20:41. doi: 10.1186/s13058-018-0966-2

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© The Author(s). 2018

Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided 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 Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

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Fig. 4 — Net1 deletion inhibits RhoA activation and myosin light chain phosphorylation in tumors. a Representative examples of staining for myosin light chain 2 phosphorylated on serine 19 (pMLC2) in genotypes shown. Only solid tumors analyzed. b Quantification of pMLC2 staining as percentage of total area in genotypes shown. Seven areas analyzed, 3 animals per genotype. c Analysis of myosin phosphatase 1 (MYPT1) phosphorylation on its activating site T696 in tumor lysates. Each lane represents lysate from a tumor from a separate animal. d Analysis of active RhoA and RhoC activation in tumor lysates using GST-Rhotekin binding domain (GST-RBD) pulldowns. Each lane represents lysate from a tumor from a separate animal. e Quantification of RhoA-GTP/total RhoA for GST-RBD pulldowns. Six tumors per genotype quantified. f Quantification of RhoC-GTP/total RhoC for GST-RBD pulldowns. Six tumors per genotype quantified. g Quantification of primary tumor cell motility toward EGF in modified Boyden chambers. Data represent mean number of migrated cells/field from three separate tumors per genotype, isolated from separate mice, performed in duplicate. Errors are standard error of the mean. *P < 0.05; ***P < 0.001. GST glutathione-S-transferase, MYPT1 myosin phosphatase targeting subunit 1, Net1 neuroepithelial transforming gene 1, n.s. not significant, PyMT polyoma middle T antigen, RBD RhoA binding domain, RhoA Ras homolog family member A, RhoC Ras homolog family member C