Figure 3. LRP5.Q89R did not affect binding to MESD or protein trafficking in HEK293T cells. LRP5.WT-MycHis, LRP5.Q89R-MycHis or LRP5.G171V was transiently expressed in HEK293T cells with (A) or without (B) MESD-FLAG by transfection. A: Co-IP of LRP5 and MESD protein complex. Input: LRP5.WT, LRP5.Q89R, LRP5G171V and MESD protein levels were studied by western immunoblotting with anti-cMyc or anti-FLAG antibodies, respectively. Whole cell lysate was immunoprecipitated with an anti-FLAG antibody (IP: anti-FLAG). The enriched protein was analyzed with SDS–PAGE and detected with anti-cMyc antibodies. B: Pull-down of cell surface LRP5 proteins. Proteins on the cell surface were labeled with biotin and pulled down with Neutravidin beads. Protein samples from input and after pull-down were subjected to western immunoblotting. LRP5.WT, LRP5.Q89R, and LRP5.G171V were probed with an anti-cMyc antibody. Experiments were performed in biologic replicates and representative data were shown.