Fig. 1. Nmyc1 is phosphorylated on conserved MB1 sites and phosphorylated Nmyc1 can be detected in the developing cerebellum. (A) Known phosphorylation sites in Myc and L-Myc. Conserved putative sites for phosphorylation of Nmyc1 within the MBI (I) region at theronine-50 (T-50) and serine-54 (S-54) are indicated. (B) Chromatographic results of mass spectrometric analysis of Nmyc1 protein overexpressed in HEK293 cells. The tryptic fragment identified as Nmyc1 K44-R57 contained two phosphorylated residues (black arrow). (Inset) The fragment Nmyc1 K44-R57 was subjected to further fragmentation and the specific amino acid residues were identified. Of these, T50 and S54 were phosphorylated. (C) Anti-phosphorylated-T58-Myc antibody recognizes Nmyc1 phosphorylated on T50. CGNP cultures, which do not express Myc (Kenney et al., 2003), were infected with the indicated retroviruses for 24 hours and protein lysates were analysed by western blot. Anti-phosphorylated-T58-Myc antibody recognized wild-type Nmyc1, but not Nmyc1 mutated at T50 or S54. Nmyc1 S54 phosphorylation is evidently required for phosphorylation of T50, consistent with previous findings for Myc (Sears et al., 2000). (D) Phosphorylated Nmyc1 proteins are present in proliferating cells of the developing mouse external granule layer (EGL). (Left) Sections of PN 7 mouse cerebella were immunostained with anti-T58-Myc antibody (red) and Calbindin (green), a marker of Purkinje neurons (Pur) underlying the EGL. Nuclei are labeled with DAPI. (Right) Staining with anti-T58-Myc antibody (red) and PCNA (green), a marker of proliferating cells, confirms co-expression (indicated by merged staining, yellow).