Figure 8
Sfrp1 NTR and Sfrp1 CRD bind to Wnt8 and Frizzled-5, respectively, antagonizing canonical signalling. (a) HEK 293T cells were transiently transfected with Wnt8-HA, Sfrp1-myc, Sfrp1CRD-myc or Sfrp1NTR-myc expression constructs. Conditioned media containing similar amount of each of the Sfrp1-myc derivates (ii) were mixed with conditioned media from Wnt8-HA (iii) or from mock transfected cells (Additional file 2). Proteins from mixed conditioned media were precipitated with a polyclonal anti-HA and blotted with a monoclonal anti-myc (i). In these conditions, both Sfrp1-myc and Sfrp1NTR-myc (red asterisks) specifically co-immunoprecipitated with Wnt8-HA, while Sfrp1CRD-myc did not. Comparable levels of Sfrp1 and its derivatives were immunoprecipitated (iv). Note that Sfrp1NTR-myc migrates as a smear due to post-translational glycosylation. Sfrp1CRD-myc likely suffers similar post-translational modifications and possibly forms dimers (arrow in (ii)) that do not completely dissociate. (b) Cells dissociated from E5 embryonic retinas were co-transfected with a reporter plasmid containing 4 × Lef-1 responsive element together with Wnt8, Fz5 ( 100 ng) in combination with the PCDNA plasmid alone (200 ng) or containing Sfrp1, Sfrp3, Sfrp1NTR, Sfrp3NTR, Netrin-1NTR or Sfrp1 CRD constructs as indicated in the graph. Wnt8/Fz5 co-transfection activated the reporter expression 140-fold. This activation was strongly inhibited by the addition of Sfrp1, Netrin-1NTR, Sfrp1NTR or the combination of Sfrp1NTR and Sfrp1CRD. Equivalent amounts of Sfrp3, Sfrp3NTR or Sfrp1CRD alone were less effective. Data represent means ± standard error from three separate experiments performed in triplicates (*p < 0.05; **p < 0.01; ***p < 0.001; Student's t-test). (c) HEK 293T cells were transiently co-transfected with plasmids encoding Sfrp1-myc, Sfrp1CRD-mycor Sfrp1NTR-myctogether with Fz5-HA expression vector (a) or PCDNA vector (Additional file 2). Proteins from cell lysates were precipitated with anti-HA and then blotted with anti-myc antibody. Note that Sfrp1 and Sfrp1CRD (red asterisks) interacted with Fz5 while the Sfrp1NTR did not. IP, immunoprecipitation; WB western blot.