Figure 7: Detection of endogenous zNMTs during early development. (A) The embryos at 2-72 hpf were homogenized in 5×SDS-PAGE sample buffer and the protein in the extracts were separated by SDS-PAGE followed by immunoblotting with antiserum against zNMT1. Alternatively, the protein extracts from 6 hpf and 72 hpf were assessed by immunoblotting with antiserum against thioredoxin (Trx) as a negative control. Arrows showed specific bands detected by the antiserum. (B) The zNMT1-myc-His protein was expressed in 6 hpf embryos and total proteins were extracted. The zNMT1-myc-His was purified with Ni-Sepharose beads; the protein was assessed by immunoblotting with anti-myc antibody. (C) The protein extracts were prepared from embryos at 6 hpf. The embryonic lysate was reacted with biotinylated substrate peptide or biotinylated N-myristoylated substrate peptide in presence or absence of myristoyl-CoA (mCoA) as indicated. The substrate peptides linked to proteins were detected by SDS-PAGE followed by immunoblotting with anti-biotin antibody (asterisks).