α tubulin monoclonal antibody

Phosphorylation of Mitogen-activated Protein Kinases (MAPKs) was analyzed in monokaryons of F- and S-subpopulations. Mycelia were obtained as described previously for RNA-seq experiments. Briefly, frozen mycelia maintained at −80 °C were ground in a mortar, and resuspended in protein extraction buffer containing 10 mM HEPES, 50 mM KCl, 1 mM EGTA, 1 mM MgCl₂, Protease Inhibitor Cocktail tablets (Roche Diagnostics, SL, Basel, Switzerland) and PhosSTOP Phosphatase Inhibitor Cocktail tablets (Roche Diagnostics, SL). Samples were centrifuged to pellet debris, and the protein concentration of the soluble fraction was measured by Bradford assay (Bio-Rad Laboratories, Inc.). Twenty-five micrograms of total protein were separated in a 10% SDS-PAGE gel and transferred to nitrocellulose membranes for Western blot analysis using a Trans-Blot Turbo Mini Nitrocellulose Transfer Pack following the manufacturer’s instructions (Bio-Rad Laboratories, Inc.). Phosphorylation of TEY and TGY motifs of MAPKs were detected with phosphor-p44/42 (ERK1/2 homologous) and -p38 (Hog1 homologous) MAP kinase antibody kits (Cell Signalling Technology, Danvers, MA, USA) following the manufacturer’s instructions. An α-tubulin monoclonal antibody (Sigma Aldrich) was used for the loading control.