Figure 8. DARPin binding to membrane-embedded LmrCD.

(A) Six DARPins (each at a 350 nM concentration) specific for AcrB or LmrCD were probed for binding to ISOVs containing either overproduced AcrB_AviC or LmrCD_AviC. Bound DARPins were detected on Western blot (left panel). The signals of the DARPin-specific bands were quantified by densitometry (right panel). Total binding denotes the quantified amount of DARPin bound to membrane vesicles containing overexpressed target protein. Background binding refers to binding to membrane vesicles containing overexpressed LmrCD_AviC in case of the AcrB DARPin 110819, or overexpressed AcrB_AviC when LmrCD-specific DARPins were used. Specific binding was calculated by subtracting background binding from total binding. (B) Binding of DARPin_Act2 and α-LmrCD#3 to ISOVs containing either overproduced AcrB_AviC or LmrCD_AviC was further assessed using increasing concentrations of DARPin (0.35 µM, 1 µM and 2 µM) and analyzed by Western blot (left panel). The data was quantified as in (A) (right panel). The data represent typical results observed in n = 3 experiments.