Assembly assay identifies a critical region of human fibrillin-1 required for 10–12 nm diameter microfibril biogenesis
Fig 3
Secretion of full length GFP-FBN1 fusion constructs from HEK293T cells.
Mutations associated with nMFS (neonatal) or cMFS (classical) were engineered into GFP-tagged FBN1 cDNA construct and transiently transfected into HEK293T cells. After 3 days of culture, samples of cells and medium fraction were analysed by Western blotting with an antibody directed against the GFP epitope. Empty vector (pcDNA) and wild-type construct (GFP-FBN1) were used as negative and positive controls. The majority of mutant constructs were easily detected in the culture medium. The C1086Y variant was consistently seen at reduced levels in the medium compared to the other constructs. Cell fraction samples indicated that expression levels were similar across the different variants and that the reduction seen for the C1086Y variant was not due to a lack of expression. *The G1127S substitution, which was identified in cases of isolated aortic aneurysm rather than cMFS, is associated with a milder form of disease and so is grouped with the cMFS variants here.