Supporting Information Figure S1 LRP6 antibodies do not bind additional cell surface proteins. To block LRP6-dependent binding to live HEK293 cells stably expressing LRP6, the indicated antibodies were incubated with different concentrations of purified LRP6 E1-E4 protein and then used for indirect immunofluorescence flow cytometry. The relative fluorescence obtained with each antibody in the absence of competing soluble LRP6 protein was normalized to 100%. Greater than 50% reduction in cell binding is achieved at a 0.62-fold molar ratio of competing protein to antibody, and over 97% of binding is blocked by 5.6-fold molar excess of soluble LRP6 protein. Found at: doi:10.1371/journal.pone.0012682.s001 (0.34 MB EPS) Figure S2 RNAi of candidate autocrine Wnts identifies isoforms mediating endogenous signaling in tumor cell lines. (A–D) Individual and pools of four siRNAs against Wnt isoforms were transfected into PA-1 (A), M14 (B), Hs578T (C), and NCI-H23 (D) cell lines stably integrated with Wnt luciferase reporter. Expression of luciferase is normalized to cell number and additionally normalized to levels in cells not transfected with siRNA. b- catenin, LRP6, and LRP5 RNAi, as well as Fzd8CRD-Fc protein (10 mg/ml) treatment, serve as controls for detection of autocrine Wnt signaling. Found at: doi:10.1371/journal.pone.0012682.s002 (0.45 MB EPS) Figure S3 Whole but not one-armed antibody crosslinks LRP6 at the cell membrane. HEK293 cells transfected for expression of both flag- and HA-tagged LRP6 were treated with the indicated antibodies, and LRP6-flag was immunoprecipitated from cell lysates. After elution, LRP6-flag complexes bound to LRP6 antibodies were affinity purified using Protein A/G resin and subjected to Western analysis for crosslinked LRP6-HA. As References 1. 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Found at: doi:10.1371/journal.pone.0012682.s003 (1.23 MB EPS) Figure S4 Apomab antibody crosslinking induces apoptosis in HT-1080 cells. We utilized the crosslinking-dependent proapop- tosis activity of Apomab antibody to optimize experimental conditions for crosslinking receptors on HT-1080 cells by either the whole IgG or F(ab9)2 fragment of anti-human-Fc antibodies (NA indicates no crosslinking antibody added). Results are shown for 1 mg/ml of all antibodies. Cell viability was monitored after 17 h of antibody treatment using the MultiTox-Fluor assay (Promega). Found at: doi:10.1371/journal.pone.0012682.s004 (0.32 MB EPS) Acknowledgments We thank Marinella Callow, Andrea Cochran, Xiangnan Du, Joshua Goldsmith, Ted Lau, David Lawrence, Elizabeth Lewis, Li Miao, Jing Qing, Chie Sakanaka, and Dan Yansura for technical assistance, reagents and advice. Author Contributions Conceived and designed the experiments: YG EB CC SS VD BYL KP TCC RADC MJS BR RNH YW PP MC. 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