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. 2010 Apr 8;5(4):e9864. doi: 10.1371/journal.pone.0009864

Figure 1. Not4p is a phosphoprotein.

Figure 1

A: Phospho-proteomics on the Ccr4-Not complex. Ccr4-Not complexes were TAP-tagged purified from a strain expressing Caf40-TAP and visualized on gradient SDS-PAGE gel by Coomassie (upper left panel), marker proteins (kDa) are indicated on the left. Tryptic digestion of Coomassie stained bands was followed by LC-MS/MS analyses, leading to the identification of the Ccr4-Not subunits and their phosphorylated peptides (inserted Table; a Phosphorylated amino acids are underlined; b Cleaved with trypsin and detected by ESI-QTOF mass spectrometry; c Cleaved with trypsin/V8 and detected by ESI-LTQ-Orbitrap mass spectrometry). A representative spectrum including peak assignment of Not4p phosphorylation on S92 is given (upper right panel; inset represents the b- and y-ion coverage of the phosphopeptide). B: Not4p is phosphorylated in vivo. TAP-tagged versions of Not1p, Not4p or Caf40p were captured on IgG beads and subjected to treatment with shrimp alkaline phosphatase (SAP) or SAP pre-incubated with phosphatase inhibitors (Inh.). Samples were resolved by SDS-PAGE and analyzed by immunoblotting using antibodies recognizing the protein A moiety of the TAP-tag (anti-PAP). Marker proteins (kDa) are indicated on the left. C: Bur1p kinase activity is not required for phosphorylation of Not4p. Strains expressing Not4-TAP and either the BUR1 or the bur1-23 allele were incubated at 37°C for the indicated hours (h). Samples were analyzed by immunoblotting with antibodies against PAP, H3K4me3 or TBP.