Total β catenin

For luciferase reporter assays, cells were harvested in 1X passive lysis buffer according to the manufacturer´s protocol (Promega, Mannheim, Germany). All samples were prepared in triplicate. TOPFLASH luciferase activity was normalized to Renilla control. All error bars shown are standard deviations from the mean of triplicates.
For western blotting, cells were harvested in 1% Triton lysis buffer (1% Triton X-100, 50 mM Tris-HCl [pH 7.0], 150 mM NaCl, 25 mM NaF, 5 mM Na₃VO₄, 5 mM EDTA and protease inhibitors). Western blot analysis was carried out following a standard protocol28 (link), by using the following antibodies: Anti-total LRP6 (T147928 (link)), PPSP phosphorylated (active) LRP6 (Sp1490, Cell Signaling Technology, Danvers, MA), total β-catenin (Sigma-Aldrich, Munich, Germany), α-tubulin (Santa Cruz Biotechnology, Heidelberg, Germany).
The secreted Dkk1-GFP fusion proteins present in the conditioned medium and used for the 2c2f lsFCS experiments were analyzed for integrity using an anti-GFP antibody (Abcam, Cambridge, UK). For the data shown in Supplementary Information, Fig. S7, 5 μl aliquots of conditioned medium were analyzed by SDS-PAGE/Western Blot.

Human osteoblasts were seeded on collagen-coated coverslips. 24 hours post seeding, HOBs were treated with a Wnt agonist, CHIR (5 nM) (Selleckchem), ABC (0.5 μg/mL), or DMSO (vehicle control) for 24 hours. Following fixation, cells were stained to assess total β-catenin (1:100, Sigma-Aldrich), hypo-phosphorylated (active) β-catenin (1:100, USBiological), anti-rabbit α-tubulin (1:100, Cell Signaling), or anti-mouse α-tubulin (1:100, Boster Bio). Detailed methods are presented in the supplement.