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. 2013 Oct 21;8(10):e79464. doi: 10.1371/journal.pone.0079464

Figure 3. CDC-7-dependent activation of DBF-2 occurs through SID-1 (A).

Figure 3

Reciprocal co-immunoprecipitation experiments of CDC-7-GFP and HA-SID-1 from cell extracts co-expressing both functionally tagged proteins indicate interaction of the two kinases. (B) In vitro DBF-2 activity assays revealed that addition of separately purified SID-1 stimulate DBF-2. SID-1-dependent stimulation of DBF-2 was further increased by addition of CDC-7 to the reaction. As control, CDC-7 alone did not stimulate DBF-2 (n = 5). To confirm equal precipitation of protein used for the kinase reactions, the kinase-sepharose pellet after the kinase reaction was boiled in Laemmli buffer and the supernatant used to determine equal protein abundance by SDS-PAGE and Western blot. (C) SID-1 was able to stimulate DBF-2, but not DBF-2(T671A). Western blot analysis of the precipitated proteins was used to determine comparable kinase levels (n = 5).