FIG 2.

miniTurbo can biotinylate proteins in human foreskin fibroblasts. (A) Western blot of whole-cell lysates. The indicated lines of HFFs were either infected or left uninfected (+/–) with RHΔhptΔku80 tachyzoites for 24 h and then incubated with or without (+/–) 50 μg of biotin for 1 h. Biotin labeling was terminated by cooling the cells to 4°C and washing away excess biotin. Whole-cell lysates (2 μg) were subsequently prepared and blotted with streptavidin-HRP to visualize biotinylated proteins and mouse anti-V5 antibodies to visualize expression of the miniTurbo fusion proteins. Bands present in the non-miniTurbo-expressing HFFs and the HFFs not incubated with biotin represent endogenously biotinylated host and parasite proteins. * denotes the location on the streptavidin blot at the approximate size of miniTurbo-RAH, and ** denotes the location on the streptavidin blot at the approximate size of miniTurbo. Approximate migration of a ladder of size standards (sizes in kDa) is indicated. (B) Representative immunofluorescence microscopy images of HFF populations stably expressing miniTurbo fusion proteins. The indicated line of HFFs was either infected with RHΔhptΔku80 tachyzoites or left uninfected for 24 h and then subjected to a 1-h incubation with biotin as described in panel A. After washing, the monolayers were fixed with methanol. The miniTurbo fusion proteins were detected with mouse anti-V5 antibodies (magenta), biotinylated proteins were detected with streptavidin-HRP (green), and the entire monolayer was visualized with phase microscopy. White arrowheads indicate a representative PV. Scale bar is 10 μm.