Fig. 6.

Phosphorylation levels of MLC2 at Ser15 are decreased. Only one phosphopeptide was detected from the TiO₂-enriched fraction of MLC2 in-gel digests, and its CID-MS2 spectrum is shown along with the determined sequence in A. Phosphorylation is indicated by “p” and oxidation is indicated by “o.” Analysis O¹⁶/O¹⁸ data indicated that the peptide bearing phospho-Ser¹⁵ decreased significantly from adult to neonatal samples, whereas the total protein load was about same, as indicated by the near 1:1 ratio of unphosphorylated peptides present in the TiO₂-unbound fraction (see representative spectra in B; additional spectra are shown in Supplemental Fig. 9). The detected lower level of phosphorylation of MLC2 at Ser¹⁵ in neonatal samples was confirmed by Western blots (C) using a total MLC2 antibody and an anti-phospho-MLC2 Ser¹⁵ antibody.