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. 2014 Sep 9;9(9):e106547. doi: 10.1371/journal.pone.0106547

Figure 7. Effect of additional mutations in the N-terminal region of the SBD on binding with PHD3.

Figure 7

(a) The 10 similar amino acids in the N terminal region (1–00) of Siah1 and Siah2 SBD are highlighted in grey. Mutated residues among the similar amino acids are highlighted within the box. (b) HEK293T cells were transfected with the expression plasmids for the indicated proteins. The cells were lysed and the cell lysates were subjected to FLAG-immunoprecipiation (IP). Immunoprecipitates and lysates were then analyzed by western blotting using the indicated antibodies. The [S1-8Mut]NT[S2]CT mutant increased binding compared to [S1-6Mut]NT[S2]CT. (c) The amount of PHD3 that coimmunoprecipitated with chimeric and mutated Siah1 and Siah2 SBD was quantified using Gel-pro analyzer software. The binding of the chimeric and mutated SBD to PHD3 was expressed as percentage of the binding of WT Siah2 SBD to PHD3. The data are represented as mean±S.E.M from three independent experiments. Differences in measured variables were assessed with Student's t test. * denotes p<0.05.