KIAA1212 binds to several proteins through its C-terminus (CT; Figure 2C), including AKT (Anai et al., 2005; Enomoto et al., 2005), Gαi/s (Le-Niculescu et al., 2005) and actin (Enomoto et al., 2005). To map the region(s) of KIAA1212 involved in association with DISC1, we generated a series of Flag-tagged N-terminal deletion mutants of KIAA1212 and co-expressed them with DISC1 in HEK293 cells (Figure S4A). Co-IP results suggested that the CT2 domain of KIAA1212 is sufficient to associate with DISC1 in the same protein complex (Figure S4B). To determine whether KIAA1212 and DISC1 directly bind to each other, we generated recombinant GST-tagged KIAA1212 fragments (Figure 2C) and performed an in vitro pull-down assay using His-tagged DISC1. Consistent with the co-IP result, the CT2 domain of KIAA1212 exhibited direct interaction with DISC1 (Figure 2D). In addition, the M1 and M2 domains of KIAA1212 also directly bind to DISC1 (Figure 2D). We further examined the affinity between the CT2 domain of KIAA1212 and DISC1 in vitro (Figure 2E). Dose-response analysis estimated a dissociation constant (Kd) at 1.0 μM, which is very similar to
