Fig 4.

Methylation and ligand-binding assays of Mlp24. (A) Methylation patterns of Mlp24 in the presence and absence of various amino acids. O395N1 cells (classical) expressing Mlp24-FLAG were incubated with (+) or without (−) serine (Ser), arginine (Arg), asparagine (Asn), proline (Pro), and glutamate (Glu), and Mlp24-FLAG was detected by immunoblotting with anti-FLAG antibody. (B and C) Binding of serine to the periplasmic fragment of Mlp24 (Mlp24p). ITC measurements of 10 μM Mlp24p were carried out with 10 mM serine (attractant [B]) and 30 mM glutamic acid (nonattractant [C]). Enthalpy changes per mole were plotted as a function of the molar ratio of serine to Mlp24p. The binding parameters calculated from the data are described in the text.