Fig. 6.

Quantification of BODIPY-prazosin free and bound components and determination of specific binding. (A) Autocorrelation analysis of BODIPY-prazosin in the presence of SMALP-ABCG2-SNAP yielded two components with differing diffusion coefficients: component C1 (‘free’) and C2 (‘bound’), respectively. Also shown are the diffusion coefficients for BODIPY-prazosin alone (green) and SNAP-Surface Alexa Fluor 647 labelled SMALP-ABCG2 (red) indicating these are comparable to the C1 and C2 respectively. Data shown are mean ± SD of 3 independent preparations, each measured in triplicate. Data were analysed by ANOVA and Tukey's multiple comparisons test and the significant difference between in diffusion co-efficient of bound BODIPY-prazosin (C2) compared to free BODIPY-prazosin (C2) is shown (**, p < 0.01). (B) BODIPY-prazosin (50 nM) was incubated with increasing concentrations of SMALP-ABCG2-SNAP (blue) or equivalent non-ABCG2-expressing SMALPs (negative control; red) for 30 min. Subsequent autocorrelation of fluorescence fluctuations allowed the concentrations of bound ligand (C2) to be determined in each sample. Data are shown as mean ± SEM, n = 3 independent preparations, each performed in triplicate. (C) SMALP-ABCG2-SNAP (100 nM) was incubated in the presence (prazosin) or absence (Ctrl) of 1 μM prazosin for 30 min prior to incubation with 50 nM BODIPY-prazosin for a further 30 min. Concentrations of bound BODIPY-prazosin were determined from autocorrelation analysis. Data were assessed for significance by unpaired t-test (** < 0.01) (mean ± SD, n = 3 independent experiments).