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. Author manuscript; available in PMC: 2007 Jan 12.
Published in final edited form as: J Biol Chem. 2006 Sep 12;281(45):34537–34548. doi: 10.1074/jbc.M606654200

FIGURE 2. Identification of phosphorylation sites on Pah1p dephosphorylated by the Nem1p-Spo7p complex.

FIGURE 2

A, hyperphosphorylated Pah1p, purified from a culture of pah1Δ nem1Δ spo7Δ expressing Pah1p-PtA by IgG-Sepharose chromatography and tobacco etch virus protease digested to remove the PtA tag was incubated with IgG-Sepharose beads with or without Nem1pPtA-Spo7p complex. Reactions were resolved by 7% SDS-PAGE followed by Coomassie staining, and the Pah1p bands were excised from gels and subjected to liquid chromatography-MS/MS analysis as described under “Experimental Procedures.” B, Pah1p-PtA purified from cells overexpressing the Nem1p-Spo7p complex using the galactose promoter or the control vectors was affinity-purified and resolved by 10% SDS-PAGE followed by Coomassie staining, and the Pah1p-PtA bands were excised from gels and subjected to nanoelectrospray mass spectrometry. In both A and B, the positions of phosphoserine residues identified by mass spectrometry within the two Pah1p samples are highlighted by asterisks (Ser/Thr-Pro sites) or triangles (non-Ser/Thr-Pro sites).