Filamentous cells were induced by spraying sporidial cells with hydroxyl fatty acids on parafilm, as described in the previous report44 (link). Parafilms with attached filamentous cells were then incubated with or without 5 µg of purified proteins in 2 ml PBS buffer (pH 7.4) for 4 h at room temperature. Subsequently, immunostaining was performed using anti-His/anti-HA antibody (Yao-Hong Biotech., Taiwan; #YH80003 and #YH80007; 1: 2000 dilution) and Alexa Fluor 488/594 (AF488/594)-conjugated secondary antibody (Invitrogen #A28175; Abcam # AB150116; 1: 2000 dilution)44 (link). For the colocalization study in AB33 filaments, the AF594-labeled filaments were stained with 1 µg/ml of wheat germ agglutinin-conjugated Alexa Fluor 488 (WGA-AF488; Invitrogen# W11261) for 10 min in a PBS buffer. The cells were then subjected to a 1-min vacuum in a PBS buffer containing 1 M NaCl before being visualized using microscopy. The fluorescence was observed using Axio Observer fluorescence microscope equipped with Axiocam 702 Monochrome camera (ZEISS, Germany). Images were processed using ZEN 3.2 imaging software (ZEISS).
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