Fusion pulse

Cells were grown to mid-log-phase with or without the following reagents: Bortezomib (BZ; A2S Cat # 2614), cycloheximide (CHX, Sigma, Cat # C6255) or cadmium (Cd; Sigma Cat # 265365). Cells were pelleted by centrifugation (3,500 × g, 5 min) and incubated with 0.1 N NaOH for 5 min, followed by centrifugation (17,000 × g, 3 min). SDS-PAGE sample buffer containing 50 mg/ml Dithiothreitol was next added, followed by boiling for 5 min. Proteins were separated on SDS-PAGE, transferred to a PVDF membrane, blocked in 10% Dry Milk in TBS + 0.1% Tween-20 (TBS-T), and then probed with primary antibodies for 1 h at room temperature. Following three washes with TBS-T, the membrane was incubated with a secondary antibody for 0.5 h at room temperature, then washed three times with TBS-T. Membranes were incubated with ECL mix (Thermo Fisher Scientific) for 2 min and reactive bands were visualized using Fusion Pulse (Vilber Lourmat).

Unless stated otherwise, 30 μg of the different lysates were separated on 12% SDS-PAGE, transferred to polyvinylidene difluoride membranes (Bio-Rad; catalog no.: 1620174) and blocked in blocking buffer, composed of Tris-buffered saline (TBS)  (10 mM Tris–HCl [pH 7.5] and 150 mM NaCl) with 0.1% Tween-20 (TBS-T) supplemented with 3% dry milk. Blots were then probed with primary antibodies diluted 1:1000 in blocking buffer overnight at 4 °C. Following three washes with TBS-T, the membranes were incubated with secondary antibody diluted 1:10,000 in blocking buffer for 1.5 h at room temperature and then washed three times with TBS-T. Membranes were incubated with ECL mix (Thermo Fisher Scientific) for 2 min, and reactive bands were visualized using Fusion Pulse (Vilber Lourmat). Antibodies used in this study are listed later (Table 3).Table 3

Antibody list used in this study

Antibody	Manufacturer
α-ERK	Cell Signaling Technology; catalog no.: 4695
α-p(TEY) ERK	Cell Signaling Technology; catalog no.: 4370
α-p(T207/188) ERK	Kinexus; catalog no.: pk865
α-His	Sigma; catalog no.: H1029
α-HA	Roche; catalog no.: 11867423001
α-Mpk1	Santa Cruz Biotechnology; catalog no.: SC6803
α-Hog1	Santa Cruz Biotechnology; catalog no.: SC9079
α-p-p38	Cell Signaling Technology; catalog no.: 9211
α-GAPDH	Thermo Fisher Scientific; catalog no.: MA515738

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.