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. Author manuscript; available in PMC: 2010 Oct 13.
Published in final edited form as: J Biol Chem. 2006 Sep 25;281(46):35316–35326. doi: 10.1074/jbc.M606568200

FIGURE 6. Interaction of Siah2 phospho-mutants with PHD3.

FIGURE 6

A, 293T cells were transfected with PHD3-FLAG, Siah2-HA, and Siah2-T24AS29A. 48 h later cells were either untreated or treated with 1% hypoxia. Cell lysates were subjected to immunoprecipitation (IP) with anti-HA antibody followed by immunoblot (IB) analysis with anti-FLAG antibody followed by anti-HA antibody. B, 293T cells were transfected with Siah-HA, double phospho-mutant Siah2 T24AS29A-HA, and PHD3-FLAG. Cells were either left untreated or treated with 1% hypoxia. Cell lysates were subjected to immunoblot analysis with anti-FLAG, anti-HA, and anti-green fluorescent protein antibodies. C, 293T cells were transfected with Siah2-HA, double phospho-mimetic mutant Siah2 T24ES29D-HA, and PHD3-FLAG. Cell lysates were subjected to immunoblot analysis with anti-FLAG, anti-HA, and anti-β-actin antibodies. D, 293T cells were transfected with Siah2-HA, Siah2 T24AS29A-HA, and Siah2 T24ES29D-HA, and PHD3-FLAG. Cell lysates were prepared after treatment with 20 µm lactacystin (4 h) or control mock treatment followed by immunoprecipitation with anti-HA antibody and immunoblot analysis using anti-FLAG and anti-HA antibodies. Cell lysates were subjected to immunoblotting and probed with anti-FLAG, anti-HA, and anti-β-actin antibodies.

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