Biochemical studies involving L-Wnt-STF or DLD-1 cells were performed in either 48- or 6-well format with IWR (10μM) or IWP (5μM) compounds and/or cycloheximide (100μM) in a 48 hr assay period. E-cadherin depletion studies were performed at 4°C using DLD-1 cells lysed in PBS/1% NP-40/protease inhibitors. For Wnt3A and ShhN phase separation assays, expression constructs encoding mPorcn, hWnt3A-myc, or mShhN were transfected into HEK293 cells using Effectene (Qiagen). After 48 hrs, cells were lysed for 15 min, RT with distilled water, 10 mM Tris-HCl, 150 mM NaCl /1% TritonX-114 (PL buffer). Lysate was briefly chilled on ice, pelleted for 10 min 4°C, and the supernatant combined with an equal volume of PL buffer/3.5% TX-114. Solutions were rotated for 15 min at 4°C, placed at 37°C for 5 min followed by an additional centrifugation for 5 min at 2000g, RT. Distinct phases were collected and combined with PL buffer to a total volume of 1 ml. Samples were chilled on ice, ConA sepharose (for Wnt protein) or 5E1 mAb/protein A sepharose (for ShhN protein) added, and samples rotated for 2 hrs at 4°C. Beads were washed 2x with PL buffer, and a Western blot performed with eluted proteins using an anti-c-myc antibody. For IWP-PB and IWR-PB binding studies, cell lysate (10mM Na2HPO4 pH7.4, 0.15mM NaCl, 1%NP-40) derived from HEK293 cells transfected with the Porcn-myc or various Axin2 constructs, or bacterially expressed Axin2 (508-761)-GST protein were incubated with either DMSO, linker (0.17 mM), IWP-3 (0.6 mM), or IWR-1 (0.2mM) for 1 hr at 4°C prior to addition of NeutrAvidin agarose resin (Pierce) and either DMSO, IWP-PB (0.17 mM) or IWR-PB (.05mM). Samples were rotated overnight at 4°C, washed with 3×10min with lysis buffer, and bound material eluted with sample loading buffer. For in vitro trypsinization studies, lysate derived from HEK293 cells expressing Axin2-myc protein and treated with or without IWR-1 was incubated at RT with 0.25 mg/ml of trypsin and 0.10 mM EDTA for indicated time periods.