Ha probe

Strains were grown in 100 mL ANM liquid medium with supplements and 10 mM ammonium tartrate at 37 °C for 16 h. For nitrogen starvation, mycelia were washed with and transferred to pre-warmed nitrogen-free ANM media for the indicated amount of time. Xylose was added to the media at a final concentration of 1% to induce PnmB and PrtA overexpression in the respective overexpression strains. Mycelia were harvested on Miracloth, pressed dried, snap-frozen in liquid nitrogen, and kept at −80 °C until protein extraction. Total proteins were extracted as previously described^{19 (link)}. In all, 50 μg of total proteins were separated in 10% SDS-PAGE gel and transferred to PVDF membrane for immuno-detection. PnmB^HA, NmrA^FLAG, and histone H3 were detected using HA-probe (Santa Cruz sc-7392), anti-FLAG® M2 (Sigma F1804), and anti-Histone H3 (Abcam ab1791) antibodies, respectively, at the concentration of 0.1 µg/mL. Goat anti-mouse (Millipore AP124P) or goat anti-rabbit horseradish peroxidase (HRP)-conjugated (Millipore AP132P) antibodies were used as the secondary antibody at the concentration of 0.1 µg/mL for Chemiluminescence detection using Clarity™ Western ECL Substrate (Bio-rad 1705060) kit.

Strains were first grown in ANM media containing supplements and 10 mM ammonium tartrate at 37 °C for 16 h, and then transferred to 10 ml nitrogen-free ANM media for an additional 4 h. For PnmB and PrtA overexpression, 1% xylose was added to the media. Mycelia were collected by filter with miracloth, and mycelia were subjected to total protein extraction as the intracellular fraction the TCA extraction method described above, while the conditioned growth media was further filtered using a 0.2 μm filter to remove all mycelia before protein extraction by a modified TCA method. Briefly, 10 μg BSA and 1 mL of ice-cold 100% TCA was added to 5 mL of filtered medium, and then 2 ml of the mixture was transferred to a microfuge tube and centrifuged at 4 °C 15000 rpm for 30 min. The protein pellet was washed with 1 mL ice-cold acetone and air-dried, followed by re-suspension in 50 μL protein loading buffer with incubation at 95 °C for 10 min. 50 μL secreted protein and 10 μg intracellular total protein was separated in 10% SDS-PAGE and subjected to western blot analysis using HA-probe (Santa Cruz sc-7392) and anti-Histone H3 (Abcam ab1791) antibodies at the concentration of 0.1 µg/mL.