Wnts Specify LRP6 mAb Activity Figure 1. LRP6 monoclonal antibodies antagonize and potentiate Wnt3a-induced signaling and teratocarcinoma autocrine Wnt signaling. (A) Six antibodies against LRP6 E3-E4 protein fragment inhibit in a concentration-dependent manner the Wnt luciferase reporter activity in HEK293 cells induced with 0.1 mg/ml purified Wnt3a. Error bars of this and all other graphs, except where noted, represent standard deviation of at least 3 replicate samples. (B) Western analysis of HEK293 cells either unstimulated (2) or induced (+) with Wnt3a and treated with the indicated LRP6 antibody or purified protein. RNAi experiments demonstrate that only the lower molecular weight band recognized by the b-catenin polyclonal antibody represents b-catenin protein (data not shown). b-actin and GAPDH protein levels are shown as sample loading controls for the upper and lower gels, respectively. (C) YW210.09 antibody potentiates Wnt reporter gene activity in a manner proportional to Wnt3a concentration in HEK293 cells (NA indicates no addition of Wnt3a or antibody). (D) Concentration-dependent inhibition and potentiation of autocrine Wnt signaling in PA-1 teratocarcinoma cells transfected with luciferase reporters and treated with LRP6 antibodies, either individually or in combination, or with Fzd8CRD- Fc protein. (E) qPCR expression analysis of Wnt-induced genes SAX1 and GAD1 and Wnt-repressed gene LEFTY2 in PA-1 cells treated with or without (NA) 0.3 mg/ml Wnt3a protein, and treated with 10 mg/ml YW211.31 antibody, anti-gD monoclonal antibody as a negative control, or Fzd8CRD-Fc protein as a positive control. doi:10.1371/journal.pone.0012682.g001 required for potentiation, since high antibody concentrations would favor monovalent interactions and thus limit crosslinking of LRP6 molecules. Treatment of PA-1 cells with a combination of both YW211.31.57 and YW210.09 antibodies antagonizes both Wnt3a-induced and autocrine Wnt signaling, similar to the effect of YW211.31.57 alone (Figure 1D). PLoS ONE | www.plosone.org 3 September 2010 | Volume 5 | Issue 9 | e12682