Figure 6.

Ser²⁴⁹⁷ is phosphorylated in the HARE CD. (A) Immunopurified WT HARE cut from an SDS-PAGE gel was trypsin digested and samples were untreated (top) or treated with alkaline phosphatase (bottom). Mass spectrometry analyses using a Voyager Elite MALDI-TOF instrument in reflector mode revealed signals at m/z 2284.98 for a phospho-(Thr²³⁸⁹-R²⁵⁰⁸) peptide, which was reduced by phosphatase treatment, resulting in strong signals for the predicted free peptide (m/z 2205.02). (B) HARE was immunopurified from stable Flp-in 293 cells expressing WT (left) or S2497A (right) HARE, run on SDS-PAGE, electroblotted, stained with Pro-Q for phosphoryl groups [57,58] and HARE (arrows) was visualized by fluorescence microscopy (top) and then, after washing and staining, with Coomassie blue (bottom).