Western Blot Analysis of GFP-tagged Aspergillus

Western blotting was carried out as previously reported (51 (link)). Briefly, 1 × 10⁶ conidia of the AfERG10A::GFP and WT strains were inoculated in liquid CM and shaken at 37°C for 48 h. The mycelia were harvested and ground in liquid nitrogen with a mortar and pestle and suspended in ice-cold extraction buffer (50 mM HEPES [pH 7.4], 137 mM KCl, 10% glycerol, 1 mM EDTA, 1 μg/ml pepstatin A, 1 μg/ml leupeptin, 1 mM phenylmethylsulfonyl fluoride [PMSF]). Ten micrograms of proteins per lane was loaded onto a 10% SDS-PAGE gel. After electrophoresis, proteins were transferred to a polyvinylidene difluoride (PVDF) membrane (Millipore) in 384 mM glycine, 50 mM Tris (pH 8.4), and 20% methanol at 250 mA for 1.5 h. The membrane was then blocked with phosphate-buffered saline (PBS) containing 5% milk and 0.1% Tween 20. The membrane was then incubated in anti-mouse GFP primary antibody (Roche) at a 1:20,000 dilution and goat anti-mouse IgG-horseradish peroxidase secondary antibody at a 1:5,000 dilution. Blots were developed using the Clarity ECL Western blotting detection reagents (Bio-Rad), and images were acquired with a Tanon 4200 chemiluminescent imaging system (Tanon).