Fig. 3.

RFPs form stable complexes with RAB25 and compete for FIP binding. a Schematic of the RAB25 ALPHAscreen proximity assay. Incubation of a synthetic, biotinylated-FIP-derived peptide with tagged (His₆- or GST-) RAB25 leads to the formation of the soluble complex and proximal association of streptavidin-coated donor beads with acceptor beads (Ni²⁺-NTA or anti-GST). Donor bead excitation at 680 nm produces singlet oxygen, which selectively initiates a luminescent cascade in bound acceptor beads. b Bio-RFP14 binds H₆- (left) and GST-RAB25 (right) in a dose-dependent manner, leading to robust ALPHAscreen signal. c Competitive inhibition of RAB25:RFP14 complex formation by soluble RFP and FIP peptide ligands. Constant H₆-RAB25 (0.2 μM) and bio-RFP14 (0.2 μM) were incubated in the presence of indicated ligand concentrations prior to addition of ALPHAscreen reagents and reading of luminescent signal. d RFP stapled peptides (1 μM) compete with HA-RAB25 binding and co-immunoprecipitation of FIP1 in HEY RAB25 cell lysate. These results are representative of experiments performed in triplicate. Binding data points represent the mean ± s.e.m. from triplicate measurements. IC₅₀ values represent mean with 95% confidence interval from triplicate replicates and application of a sigmoidal curve fit using Prism 5 software