npg Axin/TNKS/KIF3A complex regulates GLUT4 translocation 1256 differentiate to adipocytes. Mature adipocytes (8 d) were serum starved for 4 h, incubated with or without 200 nM insulin for 20 min and then subjected to a 2-deoxy-D-glucose (2-DG) (0.25 µCi 3H-2DG per well) (Perkin Elmer). 10 min later, after three times washes, cells were lysed and intracellular 2-DG, which cannot be metabolized, was measured by liquid scintillation counting. Plasma membrane isolation According to the protocol described in [43], 3T3-L1 adipocytes were collected and broken by passing through a 25-gauge needle in a hypotonic buffer (50 mM Tris-HCl, pH 8.0, 0.1% NP-40, 0.5 mM DTT, protease inhibitors and phosphatase inhibitors). After centrifugation at 1 000× g for 10 min, the supernatant was col- lected. The pellet was washed two times with hypotonic buffer without NP-40 and lysed in a buffer (50 mM Tris-HCl, pH 8.0, 1% NP-40, 0.5 mM DTT, protease inhibitors and phosphatase inhibi- tors) for 1 h at 4 ºC. The re-suspended pellet was then centrifuged at 16 000× g for 20 min. The supernatant was collected as the PM fraction. The supernatant collected from the first centrifugation was cleared by centrifugation at 16 000× g for 20 min, and the re- sulting supernatant was collected as Post-PM fraction. GTT and ITT For GTT, mice after overnight fast (16 h) were intraperitoneally injected with D-Glucose (2 g/kg), and blood glucose concentra- tions were determined at 0, 15, 30, 60, 120 min after injection. In ITT, animals that had been fasted for 4 h were administrated by intraperitoneal injection of human insulin (1 U/kg) and blood glucose levels were determined at the indicated times with Accu- check (Roche). Acknowledgments We thank Dr Chi NW for providing the CMV2-FLAG-TNKS2 (human) construct. 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