Fig 9. Identification of in vitro WOX5 phosphorylation sites.

(A) The sequence of the MBP fused WOX5 protein is shown: the N-terminal MBP fusion tag (grey italics) and the 182aa WOX5 sequence with the C-terminal 6X-His tag (black). Note, the N-terminal Met residue of WOX5 was changed to Gly. Confirmed phosphorylation sites, Ser², Ser⁴, Ser⁸⁸, and Ser¹⁵⁸ are boxed. The phosphopeptides identified by mass spectrometry are underlined. (B) Collision-induced low resolution fragmentation spectrum of the phosphopeptide GSAGAALGSFSVK, encompassing the C-terminal end of the fusion tag and the N-terminal end of WOX5. The presence of the phosphorylated b₉ (theoretical m/z 654.32, observed m/z of 654.33) and y₅ (theoretical m/z 549.30, observed m/z of 549.33) ions, and the unphosphorylated b₈ (theoretical m/z 585.30, observed m/z of 585.17) and y₄ (theoretical m/z 480.28, observed m/z of 480.30) ions, confirms Ser² as the site of phosphorylation.