Fig 2. Construction of the NterPro-TB3cbEGF19 mini-gene and secretion of cMFS and nMFS variants from fibroblasts.

A) NterPro-TB3cbEGF19 is a fusion of the N-terminal domains of FBN1 up to the proline-rich region (orange) with domains TB3 to cbEGF19, which encompass the neonatal region. Western blotting using an antibody directed against the proline-rich domain (orange) is used to distinguish the fusion construct from endogenous FBN1 expressed by fibroblasts on the basis of size. B) Model of the TB3-cbEGF19 domains highlighting the positions of the nMFS (red spheres) and cMFS (orange spheres) substitutions described in this work, and the position of domain cbEGF16, which is deleted in one of the nMFS variants. Domain TB3 is coloured blue, cbEGF domains are coloured green and Ca²⁺ ions bound to the cbEGF domains are shown as grey spheres. C) Secretion profiles of the neonatal region mutants associated with nMFS and cMFS. Medium samples from untransfected MSU-1.1 fibroblasts (MSU-1.1) and fibroblasts transfected with the wild-type (WT) construct were used as controls to allow the identification of the recombinant construct (MW ~130kDa, arrowed). Full-length FBN1, expressed endogenously by the fibroblasts, functions as a loading control (MW ~350kDa, arrowed).