Figure 2

Identification of putative FIP3 phosphorylation sites Panel A. Synchronized HeLa cells from metaphase were lysed and FIP3 immuno-precipitated (from untreated or phosphatase-treated lysates) using anti-FIP3 antibodies. Immunoprecipitates were then separated by 2D SDS-Page and immunoblotted with anti-FIP3 antibodies. Asterisks mark different phospho-states of FIP3. A representative blot is shown for each condition. Panel B. Synchronized HeLa cells in metaphase were lysed in the presence of phosphatase inhibitors. FIP3 was then precipitated and putative phosphorylation site identified by mass spectrometry. Shown are all the peptides identified for each putative phosphorylation site. Panel C. A schematic of FIP3 showing the location of key residues. Phospho-acceptor sites identified here are shown in blue, those in black at the sites mutated to A in the HA-FIP3-4A mutant (see text). The Rab11-binding region (RBD) and Arf6 interaction region (ABD) are shown, together with the approximate position of the EF-hand-like domains and the coiled-coil region.