Figure 3

Analysis of Hog1 phosphorylation after addition of iron. (A) A yeast strain expressing GFP-tagged Hog1 (Hog1-GFP) was grown until the exponential phase. Protein was extracted from cells incubated for 1 h with increasing concentrations of iron (50, 100 and 200 μM/1h). The phosphorylated form of Hog1 was detected using an anti-phospho-p38 antibody (phospho-Hog1). The western blot membranes were probed for total Hog1 using a polyclonal anti-GFP antibody and this served as a loading control (B) Cells were treated with BPS for 30 min followed by addition of iron (100 μM) and cells were harvested at indicated time points. Proteins were extracted and phosphorylation of hog1 was detected by phospho-hog1 antibody. The western blot membranes were probed for total Hog1 using a polyclonal anti-GFP antibody and this served as a loading control (C) Protein was extracted from cells incubated with 100 μM CUR at the indicated time points. The western blot membranes were probed for phospho-Hog1 and anti-GFP (Total Hog1p). The intensity of phosphorylated Hog1 was quantified using Image J software and normalized with respect to total Hog1p levels (anti-GFP) and shown in the form of bar diagramme. The error bars represent the standard deviation (SD) of three independent replicates. (D) Yeast strain expressing GFP-tagged Hog1 (Hog1-GFP) was treated with 100 μM CUR for 30 min followed by addition of iron (100 μM). Cells were harvested at indicated time points in figure and protein were extracted. The phosphorylation of hog1 was detected by phospho-hog1 antibody. The western blot membranes were probed for total Hog1 using a polyclonal anti-GFP antibody and this served as a loading control. The intensity of phosphorylated Hog1 was quantified using Image J software and normalized with respect to total Hog1p levels (anti-GFP) and shown in the form of bar diagramme. The error bars represent the standard deviation (SD) of three independent replicates. (E) Hog1 Δ cells were treated with alpha-factor to synchronize them in the G1 phase. After synchronization, cells were released into media supplemented with (a) DMSO (control), (b) 100 μM CUR, (c) 100 μM CUR supplemented with 100 μM Iron and (D) 100 μM Iron. The cultures were sampled at the indicated time points and their DNA content was then analyzed by FACS.