Figure 2

Hog1 protein is phosphorylated in response to curcumin treatment. (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 CUR (0, 5, 10, 20, 50, and 100 μM). 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) An exponentially growing wild type yeast strain was exposed to 100 μM CUR for 2 h. Samples were taken after 0, 30, 60, and 120 min of incubation. The expression of HOG1 mRNA levels was examined by SYBR Green real-time PCR. The error bars represent the standard deviation (SD) of three independent replicates. (C) Protein was extracted from cells incubated with either 0.8 M NaCl or 100 μM CUR at the indicated time points. The western blot membranes were probed for phospho-Hog1 or total Hog1 (anti-GFP). The anti-GFP signal served as a loading control.