Bzb induces β-catenin accumulation in the nucleus of OBs and MSCs. Nuclear and cytoplasmic fractions of cells of indicated cell lines (A) or msc from myeloma patients (B) described in Figure 1 were separated. Nuclear fractions were lysed and proteins isolated. A total of 5 mg of protein was immunoblotted with β-catenin antibodies followed by membrane stripping and hybridization with an antibody anti-Lamin B to control for protein loading (bottom). Arrows represent ubiquitinated β-catenin. Saos-2 (Ci-Ciii), and MG63 (Di-Diii) cells were treated with 0 (Ci,Di), 50 nM (Cii,Dii), and 100 nM (Ciii,Diii) of Bzb for 12 hours. MSCs from MM patients were cultured in the absence (Ei) or presence of 12.5 (Eii) or 50 nM (Eiii) Bzb for 12 hours. Immunofluorescence microscopy with an antibody specific for β-catenin was used to assess changes in nuclear and cytoplasmic β-catenin. For panels C through E, an Olympus IMT2 inverted research microscope (Olympus, Tokyo, Japan) using an Olympus 40×/0.65 P and a X-CITE 120 illumination systems designed especially for fluorescence microscopy. The fluorescent images were acquired using a SPOT camera (Diagnostic Instruments, Sterling Heights, MI) Model: 2.21 and were processed with an Advanced STOP version 4.7 software (Diagnostic Instruments) and Adobe Photoshop CS2 version 9.02 software (Adobe Systems, San Jose, CA). (F) The indicated cells were treated with indicated concentrations of Bzb for 6 hours. Free β-catenin protein separated using GST-E-cadherin pull down in nuclear lysates was determined by immunoblotting analysis as described in “Immunoblotting.”