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. 2015 May 29;5:10627. doi: 10.1038/srep10627

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Copyright © 2015, Macmillan Publishers Limited

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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HaCaT cells were cultured in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal calf serum and in HEC-C1 medium for the last 24 h. HAoECs were cultured in HEC-C1 medium. (a) HaCaT cells were stained with anti-MRTF-A antibody or anti-MRTF-B antibody (red), phalloidine-Alexa 488 (green), and Hoechst 33258 (blue). Representative images from at least three independent experiments are shown. (b) IB analysis shows the expression levels of MRTF-A/B and proteins involved in their nuclear import and export in HAoECs and HaCaT cells (upper panel). Whole cell lysates (WL) were subjected to IB with the indicated antibodies. α□tubulin was used as a loading control. RT-PCR analyses for monitoring the expression of MRTF-A isoforms (MAL fl [fl], variant X1 [X1], and variant X2 [X2]) in HAoECs and HaCaT cells (lower panel). PCR products were sampled at the indicated time points after 20 to 27 cycles and separated on 1.2% agarose gels. (c) Nuclear accumulation of exogenously expressed MRTF-A in HAoECs. HAoECs expressing Flag-tagged mouse MRTF-A (MAL fl) were stained with anti-Flag antibody (red) and Hoechst 33258 (blue). (d and e) IB analysis shows the expression levels of MRTF-A/B and β−actin in the whole cell extracts (WE) from HAoECs and HaCaT cells (d). The respective WE were subjected to IP analyses with a control antibody (cntl-Ab) or either anti-MRTF-A antibody (e left panel) or anti-MRTF-B antibody (e right panel). The IP/IB analyses were performed with the indicated antibodies. Positions of molecular weight markers (kDa) are indicated between the IB panels. (f) Actin fractionation. HAoECs and HaCaT cells were either left untreated (jasp-) or treated with jasplakinolide (0.3 μM; jasp+) for the last 60 min. The respective lysates (L) were separated into supernatant (S) and pellet (P) fractions by centrifugation, and they were subjected to IB with anti-β−actin antibody. (g) ERK phosphorylation of MRTF-A. WL from the indicated cells were subjected to IB with anti-MRTF-A or the phospho-specific MRTF-A antibody (left panel). WE from respective cells expressing Flag-tagged mouse MRTF-A (MAL fl) were subjected to IP/IB analysis with the indicated antibodies (right panel).