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

Figure 1.

Figure 1

Both rodent and human Par-4 interact and are substrates of CK2. (a) COS cells were co-transfected with rat GFP-tagged Par-4 or empty GFP vector together with the CK2α and/or the CK2βHA-tagged subunits. In the left panel, immunoprecipitation of GFP vector, Par-4 (GFP) or CK2α subunits (HA). In right panel, immunoprecipitation of Par-4 (GFP) or CK2α and/or CK2β subunits (HA). The immunoprecipitations were followed by immunodetection of either CK2 (HA) or Par-4 (GFP). (b) Immunoprecipitation, from a same PC-3 cells extract, of endogenous human Par-4 (hPar-4) or CK2 subunits was followed by immunodetection of endogenous human CK2α, CK2β or Par-4. Inputs: proteins in total cell lysates. IP IgG: immunoprecipitation with a non-relevant antibody (IgG mouse). *HC, LC immunoglobulin heavy chain and light chain respectively. (c) The GFP-tagged CK2 kinase or GFP alone (as a control, Mock) were immunoprecipitated from transfected COS cells and used for an in vitro kinase assay in the presence of [γ−32P]ATP with either recombinant GST-Par-4 or GST alone, as substrates (left panel). Reaction products were resolved by SDS-PAGE on 10% gels, stained with Coomassie blue to verify that equal amounts of GST-Par-4 or GST alone were used in each reaction (lower panel), and autoradiography was performed (upper panel). In parallel, the amount of GFP (Mock) or CK2 subunits immunoprecipitated was analyzed by immunoblotting (right panel). (d) Recombinant GFP-tagged human Par-4 (GFP-hPar-4) was produced in vitro by the TNT rabbit reticulocyte lysate system (RRL), and lysates were subsequently immunoprecipitated using GFP antibody. Used as substrates, the immunoprecipitates were subjected to an in vitro kinase assay using recombinant CK2 (recCK2) in the presence of [γ−32P]ATP for 30 min. The CK2 inhibitor (TBB, 1 μM) was incubated 5 min before addition of CK2 recombinant (middle panel). After migration, phosphorylated Par-4 (32P-hPar-4) and autophosphorylation of recombinant CK2β (32P- CK2β) was detected by autoradiography. The amount of recombinant GFP-Par-4 produced by RRL was checked by western blotting (input, lower panel)