Fig. 5. Immediate and sustained immobilization of HER2 (labeled by the affibody ZHER2) on the surface of BT-474 cells is mediated through DARPin–receptor interactions.

a Individual FRAP curves (no averaging) before and after addition of DARPin 6L1G, colored according to the starting time relative to DARPin addition, show immediate immobilization. b HER2 immobilization is similar after 5 min and 6 h of treatment. c FRAP experiments in cells treated with Latrunculin B in the absence of DARPins (LatB), and cells treated with LatB before (LatB, 6L1G) or after (6L1G, LatB) bipDARPin addition indicate that cytoskeleton interactions are not directly involved in HER2 immobilization upon DARPin treatment. d Pre-treatment with a HER2-specific HER2 kinase inhibitor (Selleckchem S2752, an analog of ARRY 380) does not affect immobilization, suggesting that immobilization is independent of HER2 kinase activation. e HER2 immobilization occurs at a wide range of expression levels, as indicated by FRAP measurements on HER2-overexpressing BT-474 cells and non-induced HEK cells stably transfected with HaloTagged HER2. Note that the sensitivity of the FRAP method limits the cell lines that can be investigated to those with at least intermediate expression levels (Supplementary Table 1 and Supplementary Fig. 2). Data for untreated cells (Untr.) and 6L1G in c are identical to those shown in Fig. 4c.