The α3 helix in the latency complex forms during TGFβ-1 binding. (a) Alignment of the α3 helix in bound (yellow) and unbound (blue) structures. (b) The 2Fo − Fc electron-density map of the α3 helix region main chain in apo LAP is shown as a black mesh and is contoured at 1.0σ. (c) Normally, TGFβ-1 is only secreted when bound to LAP. To test for the role of the α3 helix in TGFβ-1 binding, HEK293T cells were transfected with proline mutants. After 48 h, the cell-culture supernatant was assayed with an anti-TGFβ-1 antibody, which only recognizes TGFβ-1 when released from LAP. Acid-activated proline mutants formed the LTGFβ-1 complex at much lower levels than wild-type LTGFβ-1. *p < 0.05 and **p < 0.01 (t-test).