Fig. 2. Structural requirements for activation of Wnt signaling by Rspo2. (A) Domains of Rspo2. Signal peptide (light gray) followed by a cysteine-rich domain, a furin-like domain (black box), a TSP1 domain (gray box) and a basic C-terminus. Arrowheads indicate exon junctions. (B) Western blot analysis. An anti-AP antibody detected appropriately sized Rspo2AP fusion proteins in concentrated conditioned media. (C) Conditioned media containing the indicated Rspo2AP fusion proteins was incubated with untransfected HEK293T cells and cell bound AP activity determined. (D) Binding affinity of Rspo2AP fusion products to heparin agarose, only Rspo2AP and 212-243AP possessed a high affinity for heparin. (E) HEK293T cells co-transfected with TOPFLASH and pRL-TK were exposed to the indicated conditioned media. Firefly luciferase was normalized to Renilla luciferase activity. Activity induced in response to AP conditioned media was set to 1. Furin domain-containing constructs activated TOPFLASH. (F) Flag-tagged Lrp6 expression constructs with the indicated domains deleted were co-transfected into HEK293T cells with TOPFLASH and pRL-TK. Normalized luciferase activity in pcDNA3.1-transfected cells exposed to AP conditioned media was set to 1. Bars represent the mean±s.e.m. induction. Inset shows western blot of cell lysates from parallel wells. An anti-flag antibody detected expression of appropriately sized Lrp6 proteins.