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Fig. 1 Flow cytometry assay for the HEG1–KRIT1 FERM domain interaction. (A) Ribbon diagram of KRIT1 FERM domain in complex with the HEG1 cytoplasmic tail (PDB ID: 3u7d). The HEG1 peptide is shown in yellow. The KRIT1 FERM domain consists of three subdomains: F1 (green and blue); F2 (red); and F3 (orange). The feature of the F1 domain that is not present in other FERM domain is shown in blue and that region is an important part of the HEG1 binding pocket. (B) Schematic representation of the HEG1 cytoplasmic tail (a.a. 1274–1381) peptide coupled to Neutravidin beads and the EGFP-KRIT1 FERM domain. Binding of EGFP-KRIT1 FERM domain to the HEG1 matrix beads can be detected by flow cytometry. Small-molecule inhibitors HKi preventing the interaction of EGFP-KRIT1 FERM domain with the HEG1 matrix beads reduce the EGFP fluorescence signal. (C) Flow cytometry profile of SPHERO Neutravidin Polystyrene Particles coated with increasing amount of biotinylated HEG1 peptide and 150 nM EGFP-KRIT1 FERM domain. We noticed many beads doublets in the light scatter signal at 1,500 nM concentration of HEG1 peptide. (D) Titration curve for the interaction of EGFP-KRIT1 FERM domain with increasing amounts of HEG1 on the beads as shown in panel C, as measured by geometric mean fluorescence intensity (GMFI). We used the 150 nM HEG1 peptide concentration for future experiments. (E) Titration curve for the interaction of 150 nM HEG1 on the beads with increasing amounts of EGFP-KRIT1 FERM domain (0–250 nM) wild-type (blue line) and KRIT1(L717,721A) mutant (red line). We used the 70 nM EGFP-KRIT1 concentration for future experiments. (F) Competition binding curve of 70 nM EGFP-KRIT1 FERM domain binding to 150 nM HEG1 on the beads with increasing amounts on non-biotinylated HEG1 7-mer peptide. We used the 2 μM HEG1 7-mer concentration for future experiments.