Siah1 exhibits weak binding compared to Siah2 with PHD3. HEK293T cells were transfected in 60-mm cell culture plates for 2 days with expression plasmids for the proteins indicated at the top of each panel. ( a ) Cell lysates were subjected to HA-IP and aliquots of the cell lysates and immunoprecipitates were analyzed by western blotting with the anti-FLAG antibody. Both the Full length and Siah1 SBD did not show binding to PHD3 ( b ) The lysates were subjected to reciprocal FLAG-IP. Immunoprecipitates and aliquots of the cell lysates were analyzed by Western blotting with anti-HA and anti-FLAG antibodies. In the IP, FLAG-SBD overlaps with the IgG light chain. Compared to Siah2 SBD, only weak binding of Siah1 SBD to PHD3 was observed.