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. 2014 Jan 23;5(1):e1016. doi: 10.1038/cddis.2013.532

Figure 2.

Figure 2

Identification of the CK2 phosphorylated sites S124 and S223 in rodent Par-4. (a) Two-dimensional phosphopeptide map analysis of the phosphorylated recombinant rat GST-Par-4. GST-Par-4 was phosphorylated by recombinant CK2 in the presence of [γ−32P]ATP. After migration, band corresponding to the radiolabeled phospho-Par-4 was excised from the membrane and digested by trypsin. Tryptic fragments were purified and resolved by two-dimensional thin layer electrophoresis and ascending chromatography followed by autoradiography to visualize the phosphopeptides. The separation origin is indicated as ‘0'. Three major phosphopeptides were revealed by autoradiography: P1, P1′, and P2. (b) Transfected wild-type Par-4 (rat) or 124A223A Par-4 mutants (GFP-tagged) were immunoprecipitated from COS cells and subjected to an in vitro kinase assay in the presence of recombinant CK2 and [γ−32P]ATP. Radiolabeled Par-4 was visualized upon autoradiography. Immunoblot analysis confirmed that equal amounts of GFP-Par-4 and GFP-124A223A were immunoprecipitated from each cell lysate (inputs, lower panel)