Fig. 1.

Identification of phosphorylation sites on APJ by mass spectrometry.

A, HEK293 cells expressing WT APJ were treated with 100 nM five different peptides include apelin-36, apelin-17, apelin-13, ELA-32 and ELA-21 for 10 min. Whole cell protein was extracted and subjected to tryptic digestion, and the peptides were analyzed by mass spectrometry. Shown are typical LC-MS/MS traces that identify serine 339, serine 345, serine 348 and serine 369 as phosphorylation sites. The phosphorylated residue is highlighted in red. B, C-terminal amino acid sequence alignment of APJ residues from human, rhesus monkey, chimpanzee, dog, mouse, and rat. Agonist-induced APJ phosphorylation sites identified with LC-MS/MS are highlighted in yellow. These sites were mutated from serine to alanine. Gray shading represents transmembrane helix 7. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)