FIGURE 5. Effect of Siah2 phospho-mutants on the stabilization of HIF-1α.
A, 293T cells were transfected with PHD3-FLAG, Siah2-HA, Siah2 T24A-HA, and Siah2 S29A-HA. Cell lysates were subjected to immunoblot analysis using anti-FLAG, anti-HA, and anti-β-actin antibodies. B, HeLa cells were transfected with Siah2-HA and Siah2 double phospho-mutant T24AS29A-HA. Cells were either left untreated or treated with 5% hypoxia. Cell lysates were subjected to immunoblotting and probed with anti-HIF-1α, anti-HA, anti-PHD3, and anti-β-actin antibodies. C, HeLa cells were transfected with Siah2-HA and double phospho-mutant Siah2 T24ES29D-HA. Cells were either left untreated or treated with 5% hypoxia. Cell lysates were subjected to immunoblotting and probed with anti-HIF-1α, anti-HA, anti-PHD3, and anti-β-actin antibodies.