Bromodomain proteins are necessary for BF-specific antibody internalization.

(A) Flow cytometry measuring the amount of primary anti-VSG antibody remaining on the surface following a 5-min incubation at the indicated temperatures, washout, fixation, and incubation with secondary antibody in I-BET151-treated (right) versus control cells (left). "Secondary" (gray trace) denotes control cells stained with secondary antibody only. (B) Immunofluorescence showing the amount of primary anti-VSG antibody remaining on the surface following a 5-min incubation at the indicated temperatures, washout, fixation, and incubation with secondary antibody in I-BET151-treated versus control cells. A bright spot is detected at the flagellar pocket in control cells incubated at 4°C, whereas I-BET151-treated cells have antibody distributed over the entire surface. At 37°C, control cells have internalized almost all the primary antibody. (C) Flow cytometry showing the amount of anti-VSG3 antibody bound to the surface in cells treated with I-BET151 for 3 d, compared to control cells. (D) Anti-VSG Western blot showing that protein levels of VSG at the active expression site (ES) are unaltered in I-BET151-treated cells. Tubulin is used as a loading control. Numerical data for Fig 3 is in S3 Data.