Figure 4. Effect of the lack of the IYT motif of inactive Msg5 on MAPK trapping.

(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 MLP1-GFP was examined by determining GFP signal in the same transformants (additionally carrying pMLP1-GFP plasmid) and growth conditions as in Figure 4A. 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 msg5Δ mutant strain DD1-2D, carrying the same plasmids as in Figure 4A. 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.