For GST pull-down experiments, pGST-KIAA1212 and pET28-DISC1 were introduced into E. coli (BL21) and the fusion proteins were induced with 1 mM IPTG for 4-5 hrs. For binding assay, approximate 5 μg of GST-KIAA1212 was incubated with about the same amount of His-tagged DISC1 and protein complex was purified using glutathione-Sepharose beads. For Kd calculation, a fixed amount of His-tagged DISC1 was mixed with various amounts of GST-CT2 (0 - 10 μM) and GST pull-down was performed. Amounts of the free and bound DISC1 were quantified by Western Blot analysis. The percentage of bound DISC1 was plotted against the concentration of GST-CT, and the Kd value was calculated by the Scatchard analysis.
