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. 2016 May 19;7(5):e2230. doi: 10.1038/cddis.2016.109

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Copyright © 2016 Macmillan Publishers Limited

Cell Death and Disease is an open-access journal published by Nature Publishing Group. 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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Interfering with praja2-KSR1 complex enhances ERK signaling in ES and cancer cells. (a) Cells were pretreated for 8 h with the following synthetic peptides (1 μM): scrambled-Flag and KSR1pep-Flag, renamed as go-onERK-Flag, given its role in ERK signaling (see below). Lysates were immunoprecipitated for KSR1. The precipitates were immunoblotted for praja2 and KSR1 (upper panel). Schematic representation of the scrambled and go-onERK peptides (lower panel). (b) Quantitative analysis of the experiments shown in panel (a). The data represent a mean of two independent experiments. (c) Cells pretreated (7 h) with the synthetic peptides (1 μM) (scrambled-Flag and go-onERK-Flag) were left untreated or stimulated with EGF. Lysates were immunoblotted for phosphoERK and ERK. (d) Quantitative analysis of the experiments shown in panel (c). (e) MCF-7 cells were treated for 48 h with the scrambled-Flag peptide or with go-onERK-Flag peptide, harvested and counted. A mean of three independent experiments±S.E.M. is shown. (f) E14Tg2a mouse ESCs were grown on feeder-free, gelatin-coated plates as described⁴⁹ and were induced to differentiate into EpiSCs in fibronectin-coated dishes at a density of 2.5 × 10⁵cells/cm² in the presence of 20 ng/ml Activin A and 12 ng/ml bFGF as described.⁵⁰ Lysates from cells were immunoblotted with the indicated antibodies. (g) Undifferentiated ESCs were transfected with control (siRNAc) or SMARTpool siRNApraja2. Lysates were immunoblotted with the indicated antibodies. (h) Cells treated (1 and 7 h) with the synthetic peptides (scrambled-Flag and go-onERK-Flag-Flag) or with 12 ng/ml bFGF. Lysates were immunoblotted for the indicated antibodies. (i) ESCs were grown in undifferentiated conditions in the presence of LIF and serum (ESCs) or in EpiSC medium (containing Activin) for 48 h with bFGF (EpiSCs+bFGF) or without it (EpiSCs), in the presence of scrambled-Flag or go-onERK-Flag. Q-PCR analysis of Tbx3 and Nanog mRNAs, relative to GAPDH mRNA. The results are expressed as mean±S.E.M of three independent experiments (*P<0.05)