Hog1 antibody

Frozen mycelia from C. heterostrophus strains were ground in liquid nitrogen, and their proteins were extracted using SDS buffer containing 5% SDS, 100 mM Tris pH 8, and 10 mM DTT, heated for 5 min at 95 °C and sonicated (30 s, level 5, Microson XL-Misonic, Farmingdale, NY, USA). The extract was then centrifuged (10,000 g, 10 min, room temperature), and the supernatant was subjected to acetone precipitation. The resolved protein pellet was solubilized using a urea buffer containing 8 M urea, 100 mM ammonium bicarbonate and 10 mM DTT. The protein concentration was determined using the Bradford assay, and 25 µg from each extract was loaded onto a 10% SDS-PAGE. The transfer was performed using the e-blot protein transfer kit (GenScript, Piscataway, NJ, USA). The blots were exposed to either p-P38 antibody (Cell Signaling, Danvers, MA, USA, T180-Y182, 9211S) or Hog1 antibody (Santa Cruz Biotechnology, Dallas, TX, USA, y-215, sc-9079) in order to examine the changes in the Hog1 phosphorylation levels or the total Hog1 levels, respectively, upon exposure to different treatments (sorbitol or FA). A luminescence detection by ECL was performed in a Fusion Pulse (VILBER Lourmat, Collégien, France) imager.

For HOG1 analysis, yeast cells (S. cerevisiae, BY4742; 10⁸ cells/mL) were incubated with macelignan and 0.4 M NaCl for 1 h. After disruption of the cells, 100 µg total protein was extracted and separated with SDS-PAGE. p-HOG1 and total HOG1 were detected with p-p38 (Santa Cruz, USA; sc-17852) and Hog1 antibody (Santa Cruz; sc-6815), respectively. For MPK1 analysis, cells with 10 µg/mL of macelignan were heated to 39°C to give heat stress. After 2 h, cells were disrupted and 100 µg of total protein were loaded to SDS-PAGE. p-MPK1 was detected with p42/44 MAPK (ERK1/2) antibody (Sigma) and MPK1-FLAG was detected with anti-FLAG antibody (M2; Sigma).