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. 2015 May 13;6:336. doi: 10.3389/fpls.2015.00336

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Copyright © 2015 Bekešová, Komis, Křenek, Vyplelová, Ovečka, Luptovčiak, Illés, Kuchařová and Šamaj.

This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

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Activation of Medicago sativa SIMK by three different stresses (250 mM NaCl, 15 mM H₂O₂ and 0.8 M sorbitol, all applied for a total of 30 min). (A1,A2) All stresses induce the accumulation of phosphorylated MAPK species compared to untreated seedlings as recognized by pTEpY antibody in extracts prepared in buffer E (A1) which by Phos-Tag™ (A2) show clear SIMK activation (top arrow, pSIMK; bottom arrow, SIMK). (B1,B2) The same samples extracted with buffer B show better discrimination of pTEpY immunoreactive species in western blots following common SDS-PAGE (B1) and much better separation of pSIMK (top arrow) and SIMK (bottom arrow) in Phos-Tag™ gels (B2). Panels on the left of (A1,B1) correspond to images of the protein ladder following Ponceau S staining of the membrane. Question marks in (A1,B1) denote unknown anti-pTEpY immunoreactive species.