Comparison of LRP5 and LRP6 Figure 4. Phosphorylated LRP5 and LRP6 cytoplamic domain bind similarly and directly to Axin in vitro. (A–D) Purified GST-LRP5C and GST-LRP6C were phosphorylated in vitro at 30uC for 4 hours in the presence of recombinant GSK3 and CKI plus ATP. Total protein staining (SimplyBlue) (A) shows that phosphorylated LRP5C and LRP6C exhibit slower and smeary migration, as we previously reported for LRP6C [13]. Phosphorylation of LRP5C and LRP6C at motif A was detected via Ab1490 (B), at motif C via Ab1572 (C), and at motif E via Ab1607 (D). (E) Recombinant GSK3 can associate with LRP5C and LRP6C (but not GST) regardless of their phosphorylation status. GSK3 used to phosphorylate LRP5C and LRP6C was mostly washed away via high salt buffer prior to the in vitro Axin binding experiments. Top: GSK3 in the in vitro reaction prior to wash; middle: GSK3 post wash; bottom: LRP5C and LRP6C phosphorylation at motif E; right: two exposures of the same serial dilutions of GSK3 to show the relative amount of GSK3 bound to GST-LRP5C and GST-LRP6C. (F) Protein levels after the Axin binding experiment in (G), showing comparable amounts of GST, GST-LRP5C, and GST-LRP6C in different lanes. The protein bands around the GST size in lanes 4 to 9 likely represent partial proteolytic products. (G) Axin binds equivalently and specifically to phosphorylated LRP5C and LRP6C. Remaining recombinant GSK3 bound to LRP5C and LRP6C in the in vitro reaction is also shown. Immunoblotting was done with both anti-Myc (for Myc-tagged Axin) and anti-GSK3 antibodies. Note that Axin did not bind to unphosphorylated GST-LRP5C or –LRP6C even in the presence of LRP5/6C-bound GSK3. (H) Deletion of the entire serine/threonine-rich region in GST-LRP6CDS/T (a 14-amino acid residue deletion, Figure S1B) had minimal effects on Axin binding to phosphorylated LRP6C. doi:10.1371/journal.pone.0023537.g004 PLoS ONE | www.plosone.org 6 August 2011 | Volume 6 | Issue 8 | e23537