Figure 1.

Tandem affinity purification analysis of the EWS-Fli-1-interacting proteins. (A) Tandem affinity purification procedure. 293T cells were transfected with FLAG-His-EWS-Fli-1. Forty-eight hours after transfection, FLAG-His-EWS-Fli-1 and its interacting proteins were isolated by nickel affinity chromatography followed by anti-FLAG immunoprecipitation and the protein sample was analyzed by tandem mass spectrometry. (B) EWS peptides assigned with high confidence. (C) RNA helicase A peptides assigned with high confidence. (D) FLAG-His-EWS-Fli-1 is mostly insoluble under the lysis conditions used for tandem affinity purification. The abundance of FLAG-His-EWS-Fli-1 in whole cell lysate (lane 1 and 4), tandem affinity purification lysate (lane 2 and 5), and postlysis pellet (lane 3 and 6) was determined by anti-FLAG immunoblotting. Tubulin serves as a loading control.