Figure 4: Nrp1 is cleaved by ADAM10 and ADAM17.

(a) HEK-293T cells were transfected with plasmids encoding Nrp1, Flag-tagged on its extracellular (N-terminal) end. Cultures were supplemented with TAPI-1 (20 μM), GI254023X (5 μM) or DMSO (carrier). After 24 h incubation, supernatants (sup) were analysed by western blot with antibodies against the Flag epitope for the presence of Nrp1’s ectodomain. (b) Flag-Nrp1 and shRNA constructs targeting ADAM10, or scramble shRNA as control, were co-transfected into HEK293T cells. Nrp1 levels both in the supernatants and lysates were analysed by western blot against the Flag epitope. shRNA silencing efficiency was validated by examining the lysates for ADAM10 expression levels. Total protein coomassie staining of both the sup and the lysate served as loading control. (c) Flag-tagged Nrp1 and five Myc-tagged ADAMs, or an empty vector as control, were co-transfected into HEK293T cells. Supernatants and cell lysates were analysed by western blot with antibodies against the Flag epitope, the Myc epitope and actin, as indicated. The constitutive (ADAM10) cleavage product is indicated by an arrow. ADAM17 cleavage product is marked by an arrowhead. In the anti-Myc (ADAMs) immunoblot, where two bands appear in a single lane, the upper band represents the ADAM pro-protein, while the lower indicates the mature and active MP. (d) Extracts of E13, E14 and E15 proprioceptive axons obtained from DRG insert cultures were analysed by western blot with antibodies against ADAM10 (middle) and ADAM17 (up). Anti-tubulin βIII (neuronal specific) immunoblot (down) served as a loading control. Molecular weight markers (kDa) are indicated on the right. Full-scanned western blots are presented in Supplementary Fig. 7.