D. in vitro phosphorylation assay of ATOH1. GST-tagged full length ATOH1 (FL), N-terminus ATOH1 including the bHLH domain (N) and C-terminus of ATOH1 downstream of the bHLH domain (C) were purified from N2a cells using glutathione-beads. ATOH1 proteins were incubated with either Casaine kinase 1α (CK1α) or CCND1 and CDK4 in the presence of [γ-32P]-ATP. Phosphorylation signals detected with FL, N and C by the phosphorylation of CK1α (lanes 1, 2, 3) are indicated by white arrowheads. Phosphorylation signals were also detected with FL and C by the phosphorylation of CCND1-CDK4 (lanes 5, 6, 7). Phosphorylation signals of N with CCND1-CDK4 were not detected. Strong CK1α self-phosphorylation signals were observed (lane 4). 