Phosphor p44 42

WP were supplied by China National Research Institute of Food & Fermentation Industries (Beijing, China). The molecular weights of the WP were 140−1,000 Da and accounted for 92% of the total prepared WP and included a 3−6 amino acid sequence, prepared by hydrolysis of papain method. WP consist of 98.3% protein, 0.05% lipid, 4.56% ash content, and 4.21% water.
The TRIZOL Reagent Kit of superoxide dismutase (SOD), malondialdehyde (MDA), catalase (CAT), glutathione peroxidase (GSH-PX), epidermal growth factor (EGF), interferon-γ (IFN-γ), total antioxidant capacity (TAOC), Aminopeptidase N (APN), interleukin 1β (IL-1β), transforming growth factor-β (TGF-β), tumor necrosis factor-alpha(TNF-α), interleukin-6 (IL-6), zonula occluden (ZO-1), junctional adhesion molecule (JAM-A), and intercellular cell adhesion molecule-1 (ICAM-1) were purchased from Kiel biological technology Co.(Shanghai, China). Primary antibodies against TLR4, Myd88, p38MAPK, phosphor-p38MAPK, p44/42, and phosphor-p44/42 were purchased from Cell Signaling (Beverly, MA, USA). Horseradish peroxidase conjugated secondary antibodies and β-actin were purchased from Proteintech.

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.