Protein extraction and Western blotting.

Mycelia from four 24-h stationary liquid AMM cultures in 12-well plates (1 × 10⁶ spores/ml) were pooled and transferred to either fresh AMM or AMM with 1 M NaCl for 15 min and then flash frozen, lyophilized overnight, and bead beaten for 1 min with 2.3-mm beads. Protein was extracted using the extraction buffer detailed by Bruder Nascimento et al. (96) (per 10 ml: 10% [vol/vol] glycerol, 0.1% [vol/vol] SDS, 1% [vol/vol] Triton X-100, 50 mM Tris-HCl [pH 7.5], 150 mM NaCl, 5 mM EDTA, 15 mM EGTA, 100 μl HALT protease inhibitor cocktail [Thermo Scientific], 1 mM phenylmethylsulfonyl fluoride [PMSF], 50 mM sodium fluoride, 5 mM sodium pyrophosphate, 5 mM sodium orthovanadate, 50 mM β-glycerophosphate, ddH₂O to 10 ml). One milliliter of buffer was added to powdered mycelia and vortexed, and tubes were centrifuged at 13,000 rpm for 5 min. Approximately 500 μl was removed, and protein was quantified via Bradford analysis. Forty micrograms of protein was used for each sample for Western blot analysis. Proteins were transferred from a 10% SDS-PAGE gel onto a nitrocellulose membrane for a Western blot assay using the Trans-Blot turbo transfer system (Bio-Rad, Hercules CA). Phosphorylated SakA (P-SakA) was detected using a rabbit anti-P-p38 antibody (number 9215; Cell Signaling Technology, Danvers, MA) at 1:1,000 dilution. SakA-FLAG was detected using the monoclonal anti-FLAG M2 antibody (F3165; Sigma-Aldrich) at 1:2,000 dilution. Fluorescence imaging and quantification were performed using the LI-COR Odyssey CLx system and the manufacturer’s pan/phospho protocol, along with REVERT total protein stain for normalization (LI-COR Biosciences, Lincoln, NE).