FIG 3.

The Gα protein GpaA and the G protein-coupled receptor GprM are important for repressing melanin production. (A) A. fumigatus conidia from the reported strains were inoculated into liquid MM for 72 h at 37°C. (B) Split-ubiquitin yeast two-hybrid screen with A. fumigatus GprM as the bait and the three Gα proteins as preys. Two other yeast proteins, Gap1 and Sui2, previously shown as interacting by using a split-ubiquitin yeast two-hybrid screen, were used as positive controls (41). (C) Coimmunoprecipitation (co-IP) was performed with total protein extracted from the A. fumigatus wild-type strain (lanes 1 and 4), A. fumigatus expressing a dicistronic gene including the gfp- and the HA-tagged gprM (lanes 2 and 5), and A. fumigatus expressing a dicistronic gene including the gfp-tagged gpaA and the HA-tagged gprM (lanes 3 and 6). Co-IP performed with an anti-GFP antibody specifically isolates the GprM-3×HA protein only in the presence of the GFP-tagged GpaA (left, lane 3). Similarly, co-IP performed using an anti-HA antibody specifically isolates the GpaA-GFP protein, but not GFP alone (right, lane 3). The experiment also shows that the anti-GFP antibody used recognizes an unspecific band detectable also when the beads are not attached to any protein (right, lane B). However, because of the difference in size, a clear band showing the expressed GpaA-GFP protein was detected (right, lane 3). Lanes M, SeeBlue plus2 marker (ThermoFisher).