CUR-induces Hog1 phosphorylation dependent over-expression GPD1 mRNA. (A) Nuclear-cytoplasmic extracts were made as described in materials and methods. Whole cell extract (WCE), cytoplasmic fraction (CYT) and nuclear fraction (NUC) were loaded on SDS-PAGE and transferred onto nitrocellulose membrane. Western blot with anti-H3K36me3 antibody was performed to ensure the integrity of extract fractionation (H3K36me3 is a nuclear protein). The distribution of phosphorylated Hog1 was analyzed by probing with phospho-hog1 antibody. (B) An exponentially growing wild type or hog1Δ 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 GPD1 mRNA levels in wild type or hog1Δ cells was examined by SYBR Green real-time PCR. The fold-change in GPD1 mRNA levels was normalized to the reference gene ALG9. The error bars represent the standard deviation (SD) of three independent replicates.