FIG. 6.
In vitro and in vivo interactions of Sina and Siah proteins. (A) Wild-type and mutant Siah-1 proteins bind to DCC in vitro. In the upper panel, the products of coupled in vitro transcription and translation (IVTT) of wild-type and mutant SIAH1 cDNAs are shown. Essentially equivalent amounts of [35S]methinonine-labeled wild-type and mutated Siah-1 proteins were generated, and the proteins are Flag-Siah-1 (lane 1), Flag-Siah-1-dN (lane 2), Flag-Siah-1-dR (lane 3), Flag-Siah-1(Y152) (lane 4), Flag-Siah-1(Y202) (lane 5), Flag-Siah-1(R211) (lane 6), and a luciferase control (lane 7). In the lower panel, the in vitro binding of these proteins to a recombinant GST-DCC protein (lanes 1 to 8) is shown. In lane 8, the absence of binding of the Flag-Siah-1 protein to a control GST protein is shown. (B) In vitro binding of Siah-1 to the Sina protein. Wild-type and mutated Siah-1 proteins as well as a control luciferase protein were synthesized in vitro and are shown in the upper panel. The in vitro binding assay was carried out with a recombinant GST protein containing full Sina sequences (lower panel). (C) Siah-1 and Sina proteins form homo- and heterooligomers in cells. 293 cells were contransfected with expression constructs containing cDNAs for Flag-Siah-1-dR (Flag, amino acids [aa] 2 to 38 and 77 to 282), Flag-Siah-1-dN (Flag, aa 77 to 282), HA-Siah-1-dR (HA, aa 2 to 38 and 77 to 282), Myc-Sina (c-Myc-tagged full-length Sina), Myc-Sina-dC (Myc aa 2 to 199), or control expression vector (pcDNA3), as indicated. Forty-eight hours after transfection, cell lysates were prepared and a portion of lysates was used for immunoprecipitation with anti-Flag M2 monoclonal antibody. The cell lysates (Lysates) and immunoprecipitates (IPs) were electrophoresed, and Western blotting studies were carried out with anti-Flag M2, anti-HA 12CA5, and anti-c-Myc 9E10.2 monoclonal antibodies, respectively. Flag-tagged Siah-1 proteins coprecipitated with HA-tagged Siah-1-dR (lanes 7 and 8) as well as c-Myc-tagged Sina protein (lane 9). However, the Flag-tagged Siah-1 protein did not coprecipitate with a C-terminal truncated form of Sina (lane 10). The migration positions (in kilodaltons) of selected molecular mass markers are indicated to the left of the blots. IgG, immunoglobulin heavy chain.