First of all, we transfect FLAG-PNPO and HA-DVL3 plasmids together into 293T cells. After 48h, we harvest them with IP lysate. First day, we used special antibody to connect with high-affinity magnetic beads overnight. Second day, the beads is cleanded and then connecte with protein IP lysate, whose connection is 2 mg/mL, overnight. Last day, we use elution buffer to elution binding protein and then conduct western blotting test. Next, we use Coomassie brilliant blue to stain the SDS-page gel two hours and then clean the gels with scrubbing solution overnight in roommate temperature. We cut the lanes and send to company to analysis the bingding protein with mass specture. Based on these rusults, We find different oxidation site between DVL3 wildtype and mutation.