Anti ror2

Proteins were extracted from the OS tissues or cultured OS cells by RIPA lysis buffer (Abcam) with protease inhibitor supplement. The concentration of protein in each sample was quantified by BCA assay with a standard assay kit (Thermo Fisher Scientific). Equal amount of protein was loaded for SDS‐PAGE. Proteins in the gels were then transferred to Nitrocellulose membranes (Millipore). Following that, 5% skimmed milk was used as blocking buffer to block the membranes for 30 min at room temperature and then primary antibodies were added for incubation at 4°C overnight. The membranes were washed with TBST three times and then incubated with specific species secondary antibodies (KPL) at room temperature for 2 hours. The membranes were washed again before visualization using an ECL kit. The primary antibodies used for the study were as follows: anti–GLUT1 (1:1000; Santa Cruz); anti–HK2 (1:500; Cell Signaling); (1:800; Cell Signaling); anti–LDHA (1:800; Cell Signaling); anti–Wnt5a (1:800; Abcam); anti–Ror2 (1:800; Abcam); and anti–β‐actin (1:2000; Abcam).