Figure 4.

Self-association of EWS/WT1. (A) Retention of EWS/WT1 by GST-EWS/WT1 recombinant protein. In vitro-transcribed and -translated ³⁵S-methionine-labeled EWS/WT1 was incubated with either ≈2 μg of GST (lane 2) or GST-EWS/WT1 (lane 3) bound to glutathione-Sepharose beads. Approximately 20% of the ³⁵S-methionine-labeled protein used in the experiment was loaded in lane 1 as a reference. The EWS/WT1 proteins were eluted with SDS loading buffer and analyzed by 10% SDS/PAGE. The position of the molecular mass markers are indicated on the left (NEB broad range markers) and the position of EWS/WT1 is indicated to the right. (B) EWS/WT1 self-association in vivo. 293T cells were transfected with pcDNA3:HA-EWS/WT1 and either pcDNA3:GST (lane 1) or pcDNA3:GST-EWS/WT1 (lane 2), or 293T cells were transfected with only pcDNA3:GST-EWS/WT1 (lane 3). Affinity-precipitations were performed with glutathione-Sepharose 4B beads and analyzed for the presence or absence of HA-EWS/WT1 by Western blotting with an anti-HA antibody (12CA5). + or − indicate the presence or absence, respectively, of the indicated expression vectors in the transfection mixture. The positions of molecular mass markers are indicated on the left (NEB broad range markers) and the position of HA-EWS/WT1 is indicated by an arrow. (C) The EWS/WT1 self-association domain resides between amino acids 185 and 310. A schematic diagram illustrating the EWS/WT1 deletion mutants used in this study is presented. The results from in vitro affinity selection experiments are summarized to the right. + or − indicates whether or not ³⁵S-methionine-labeled full-length EWS/WT1 could be retained on an affinity matrix containing the corresponding GST-EWS/WT1 derivative. The amino acid numbering refers to the position of the amino acids in the fusion protein, not the numbering normally found in EWS or WT1.