Protein Extraction and Western Blot Analysis

Protein extraction and western blot analysis were performed as previously described^{2 (link)}. The A. nidulans cultures were ground using liquid nitrogen, and cells were resuspended in protein extraction buffer (50 mM Tris-HCl, pH 8, 150 mM NaCl, 1 mM EDTA, and 1% NP-40) with 2 mM phenylmethylsulfonyl fluoride (PMSF), 10 mM sodium fluoride, and 1 mM sodium vanadate. The supernatant was obtained after centrifugation at 15,000 × g at 4 °C for 30 min. Total protein samples were electrophoresed on 8% SDS-PAGE and subsequently electroblotted onto Hybond-P polyvinylidene difluoride (PVDF) membranes (GE Healthcare). The membrane was blocked with 5% skimmed milk, and protein detection was carried out using anti-FLAG (Sigma-Aldrich) and goat anti-mouse IgG-HRP (Santa Cruz Biotechnology) secondary antibody following the manufacturers’ protocols (ELPISBIO). SDS-PAGE and silver staining kit (ELPISBIO) were used for silver staining.