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Figure 2. (A) Illustration of the FRET assay for the integrin conformation. The integrin ? subunit cytoplasmic tail was tagged with Aqm as a FRET donor, and the ? subunit was tagged with mCit as a FRET acceptor. When the cytoplasmic tails separate during integrin activation, FRET should be reduced. The locations of the ?GAAXR mutations (orange star) and the ?3NIN333T mutation (purple star) are indicated. (B) Heatmap of the fluorescence lifetime of integrin ?5-Aqm co-expressed with ?1-mCit (denoted ?5?1). The raw image and lifetime distribution are shown in Figures S1C and S1D. Warm colors on the SB represent longer donor lifetimes, indicating weaker FRET and the active conformation. (C) FRET efficiency (EFRET) of different integrin heterodimers. Sample size is the same as in Figure 1M, except ?V?1, n = 20 measurements (12 embryos). (D?K) Activatable integrin alleles: ?5GAAKR-Aqm co-expressed with ?1-mCit (D), ?5GAAKR?1-BiFC (E), ?VGAANR-Aqm co-expressed with ?3-mCit (F), ?VGAANR?3-BiFC (G), ?V-Aqm co-expressed with N-glycan wedge allele ?3NIN333T-mCit (H), ?VGAANR-Aqm co-expressed with ?5-mCit (I), ?VGAANR-Aqm co-expressed with ?6-mCit (J), and ?VGAANR?6-BiFC (K). White arrows in (D), (F), and (J) indicate clustering on the somite border. Scale bars, 30??m. (L) Clustering quantification of activatable integrin alleles by the SB/MC intensity ratio. ?VGAANR?3-BiFC, n = 16 (9 embryos); ?V?3NIN333T, n = 21 (15 embryos); ?VGAANR?5, n = 15 (8 embryos); ?VGAANR?6-BiFC, n = 17 (8 embryos). ?5GAAKR?1-BiFC clustering cannot be measured because of the poor membrane expression in the mesenchyme. (M) EFRET of activatable integrin alleles. Sample size is the same as in (L), except ?5GAAKR?1, n = 18 (9 embryos); ?VGAANR?3, n = 14 (9 embryos); and ?VGAANR?6, n = 15 (7 embryos). EFRET of ?V?1 (C), ?5GAAKR?1, ?VGAANR?3, and ?VGAANR?6 (M) cannot be measured in the MC because of the poor membrane expression. (C and M) Data are mean ± SD. ???p < 0.0001, two-sided t test. All experiments are in MZ?5?/? embryos.