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

Figure 6. Effect of mutations in the N-terminal region of the SBD on binding with PHD3.

Figure 6

HEK293T cells were transfected with the expression plasmids for the proteins indicated at the top of each panel. 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. (a) Both [S1-(P21H/A26P/Q62A)]NT[S2]CT and [S1-6Mut]NT[S2]CT chimeras only partially regained binding to PHD3, compared to binding of wild type Siah2 SBD to PHD3. (b) Only the chimera with the P21H/A26P mutations regained partial binding with PHD3. In contrast, mutation of Q62A did not increase PHD3 binding. FLAG-SBD Siah in the IP was masked by the IgG light chain.