previously described [28]. MEFS were generated from E13.5 mouse embryos (Lrp5 [23] and Lrp6 [7]). Individual cell lines were established and immortalized using large T antigen. No detergents were present in the lysis buffer for analysis of cytosolic b-catenin levels (610153, BD) with b-tubulin (E7, DSHB) as a loading control. MutliGauge analytic software (Fujifilm) was used to calculate cytoplasmic b-catenin levels normalized to b-tubulin. Total cell lysates were used to detect endogenous LRP5 (D80F2, Cell Signaling) and LRP6 (C47E12, Cell Signaling) in MEFS. GSK3 was detected using a monoclonal antibody (4G-1E, Millipore) and Axin-Myc was detected using either a monoclonal anti-Myc antibody (9E10, Santa Cruz) or polyclonal anti-Axin antibody (34–5900, Invitrogen) [13]. Phospho-specific LRP6 antibodies to the A site (Ab1490, Cell Signaling), C and E sites (Ab1572 and Ab1607, previously described [28]) were used to verify in vitro phosphorylation of GST-LRP5C and GST-LRP6C. Total protein was detected using SimplyBlue (Invitrogen) or Ponceau S (Sigma). Supporting Information Figure S1 Location of VSVG epitope tag and alignment of the LRP5 and LRP6 cytoplasmic domains. (A) The predicted signal peptide cleavage site for LRP5 and LRP6. Insertion of a VSVG epitope tag and BspEI site is a few amino acid residues after the cleavage site. Underlined residues represent the beginning of the first YWTD b-propeller structure. (B) ClustalW alignment of LRP5 and LRP6 proteins from human, chicken, frog, and zebrafish. Amino acid residues highlighted in black, grey, and light grey represent identical, conservative, and similar amino acids, respectively. Green boxes indicate PPPSPxS motifs A, B, C, D and E. The conserved PstI site located after motif B in LRP5 and LRP6 cDNAs was used to create the LRPAB/CDE swap constructs. The extra amino acid residues in LRP6 between the PPPSPxS motifs compared to LRP5 in gaps 1–4 are highlighted in yellow. 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