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Figure 3. (A) Integrins and ECM proteins co-immunoprecipitated with integrins ?5, ?V, or ?V?3 identified via mass spectroscopy. The intensity-based absolute quantification (iBAQ) from each replicate is color coded to show relative protein abundance. Hierarchical cluster analysis is shown as the dendrogram (see Tables S2 and S3 for protein names). Note that basement membrane ligand laminins (lama1, lamb1a, lamc1) are roughly equal in all three datasets, while thrombospondins (thbs3b, thbs4b) and cartilage oligomeric matrix protein (comp/thbs5) are found exclusively in the ?V dataset. ctl, control (FLAG-tagged myristoylated membrane-anchored GFP [mem-GFP]). (B and C) Integrin ? subunits (B) and Fn (C) quantification using median-normalized iBAQ (miBAQ). Bar indicates mean ± SD, n = 3. (D?G) Somite localization of integrin ?5?1 (D) and ?V?1-BiFC (E) in Fn double-mutant Fn?/? (fn1a?/?;fn1b?/?) embryos, ligand binding-deficient ?5FYLDD?1 in MZ?5?/? embryos (F), and ?V?3 in heat shock promoter-driven Fn1a-mKikumi transgenic (hsp70:fn1a) embryos (G). Scale bars, 30 ?m. (H and I) Clustering quantification (H) and EFRET (I) of ?5?1 in the absence of Fn, ?5FYLDD ?1 in MZ?5?/? embryos, and ?V?3 exposed to extra Fn1a. Fn?/?: ?5?1, n = 20 measurements (9 embryos); Fn?/?: ?V?1-BiFC, n = 19 (11 embryos); MZ?5?/?: ?5FYLDD ?1, n = 16 (8 embryos); hsp70:fn1a; ?V?3, n = 15 (7 embryos). Data are mean ± SD.