Anti hog1 antibody y 215

Protein extracts were prepared from mid-exponential phase cells as described previously^{15 (link)} and 50 µg of extract was resolved by SDS-PAGE on 10% gels. Phosphorylated Hog1 was detected by western blot analysis with an anti-phospho-p38 antibody (#9211, Cell Signalling Technology) as described previously^{15 (link)}. Blots were stripped and total levels of Hog1 were determined by probing with an anti-Hog1 antibody (y-215, Santa Cruz Biotechnology). Phosphorylated Cek1 and Mkc1 was detected by western blot analysis with an anti-phospho-p42/44 antibody (#4370, Cell Signaling Technology), and protein loading determined using an anti-tubulin antibody (DSHB, University of Iowa). The data are representative of three independent experiments, all of which showed similar effects.

The dually phosphorylated Hog1 in the S. cerevisiae strain was detected by western blotting as previously described [19 (link)]. Cells were treated with 25 µg/mL fludioxonil for various time periods ranging from 5 min to 3 h. The total cell extract (approx. 20 µg of protein) from each sample was blotted onto the nitrocellulose membrane. The dually phosphorylated Hog1 was detected using an anti-dually phosphorylated p38 antibody (Cell Signaling Technology, Danvers, MA, USA). The level of Hog1 was seen in the same blot after re-probing with anti-Hog1 antibody (Y-215, Santa Cruz Biotechnology, Dallas, TX, USA). For detecting phosphorylated Mpk1, anti-phospho-p44/42 MAPK (Thr202/Tyr204) (Rabbit) antibody was used (Cell Signaling Technology, Danvers, MA, USA).