Effect of the lack of the IYT motif of inactive Msg5 on MAPK trapping.

<p>(A) Western blotting analysis of the wild type strain YPH499 bearing YEp352GST (vector), YEp352MSG5GST (Msg5), YEp352MSG5^C319AGST (Msg5^C319A) or YEp352MSG5^C319A-IAYATAGST (Msg5^C319A-IAYATA) plasmids. Cells were grown to mid-log phase at 24°C and then aliquots were treated or not with Congo red (30 µg/ml) for 180 min. Protein extracts were prepared and phosphorylated MAPKs (Slt2, Kss1 and Fus3), Msg5-GST and G6PDH (as a loading control) were detected with anti-phospho-p42/44 (two upper panels), anti-GST antibody (middle panel) and anti-G6PDH antibodies (lower panel), respectively. (B) Expression of <i>MLP1-GFP</i> was examined by determining GFP signal in the same transformants (additionally carrying <i>pMLP1</i>-<i>GFP</i> plasmid) and growth conditions as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0085390#pone-0085390-g004" target="_blank">Figure 4A</a>. Data shown are the average of three independent experiments performed in duplicate. Error bars indicate standard deviations. (C) Upper panels: Western blotting analysis of the <i>msg5Δ</i> mutant strain DD1-2D, carrying the same plasmids as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0085390#pone-0085390-g004" target="_blank">Figure 4A</a>. Lower panels: sensitivity to Congo red of the same transformants as above. Cells were grown in liquid selective medium at 24°C, and a 10-fold dilution series of this culture was spotted onto YPD agar medium in the absence (no stress) or presence of Congo Red (75 µg/ml) and incubated for 2 days at 24°C. Similar results were obtained in three different experiments and selected images correspond to representative blots.