Figure 3. Gas2l3 is a Cdk1 target.

(A) The phosphorylation of Securin, Tome-1 (positive controls), and Gas2l3 was assayed in 293 late mitotic extracts [see (B) for details] in the presence of the Cdk1 inhibitors RO-3306 or roscovitine (+), or DMSO (-). Because Securin is naturally degraded in this extract system, Securin phosphorylation was assayed in the presence of MG132. (B) 293 cells were cotransfected with Myc-tagged Gas2l3 and either non-degradable Cyclin B1 or empty vector to generate asynchronous (US) and late mitotic (late M) cell populations overexpressing Gas2l3. The cells were harvested after 24 hrs for Western blotting (with the depicted antibodies) and analysis by liquid chromatography-mass spectrometry (LC-MS/MS). The samples were separated by SDS-PAGE and the area of interest, as determined by Western blotting, was excised for in-gel digestion following established protocols. The resulting peptide mixture was analyzed using a Q Exactive mass spectrometer (Thermo Scientific) equipped with a nanoflow UPLC system (Eksigent). The LC-MS data were searched against a human protein sequence database (Uniprot/Swissprot, canonical and isoform sequence data) using ProteinPilot (v4.5, AB Sciex), allowing for biological modifications/phosphorylation emphasis. Only peptide-spectrum matches above a 1% FDR cut-off were considered for further analysis. The amino acid sequence of two phosphopeptides is depicted.