FIGURE 2.

Epitope mapping reveals Ser(P)-376 (for hR3F2 and hRB7) and Ser(P)-277 (for R3G3) as phospho-targets. A, schematic depiction of known and putative phosphorylation sites and used His-tagged mutants. B–E, phospho-epitope mapping. Purified full-length His-tagged GRASP65 constructs (either wild type or phosphorylation-deficient mutants) were incubated with interphase (B) or mitotic (C–E) cell extracts, loaded in equal amounts onto separate gels, resolved with SDS-PAGE, and transferred onto nitrocellulose. Loss of signal for a given construct indicates that at least one of the mutated phospho-residues must be present as wild type and be phosphorylated for antibody binding.