Figure 3
Western blot analysis of TurboID activity in recombinant S. macrospora strains. The activity of free TurboID and SCI1-TurboID fusion proteins in S. macrospora was determined by Western blot-like hybridization using Streptavidin-HRP. Streptavidin binds biotinylated proteins, and HRP is used for signal detection via chemiluminescence. Expression of the constructs is controlled by either the ccg1 overexpression promoter from N. crassa (pc), the native sci1 promoter (p5’), or the xylose inducible Smxyl promoter (px)19. Untransformed wt shows endogenous biotinylation of S. macrospora. 2.25 µL crude protein extract were loaded onto the gel. Ponceau red staining was used as loading control. A) Strains were grown in liquid BMM medium for 3 days at 27 °C. Before harvest, BMM supplemented with biotin was added (final concentration = 410 nM) for 10 or 30 min. B) Strains were grown in liquid SWG medium (1 g/L arginine, without biotin) for 5 days at 27 °C. Shortly before harvest, SWG supplemented with biotin was added (final concentration = 410 nM) for 10 or 30 min. Blots and Ponceau red staining in (A,B) show the identical membrane, exposure was identical for all parts of the gel. Figure shows cropped blots. Full length blots and Ponceau red staining are depicted in Supplementary Fig. S6. ect, ectopically integrated.
