Fig. 2. In vitro verification of the Rab22a-EEA1 interaction. GST-Rab5a and Rab22a fusion proteins, or GST as a negative control, were bound to glutathione sepharose beads, loaded with either GDP or GTPS and incubated with a MBP-EEA1 (aa 1-209) fusion protein as descibed in the Materials and Methods. The binding of MBP-EEA1 was visualized by SDS-PAGE and western blotting with anti-EEA1 antibodies (the top panels). Specific binding of MBP-EEA1 to the GTPS-bound forms of both Rab5a and Rab22a was observed; the signal for the GDP-loaded proteins was very weak. Background binding of MBP-EEA1 to plain GST was undetectable. Visualization of the fusion proteins on the filters by Ponceau staining (the bottom panels) reveals that the amount of Rab22a required for a given signal intensity is markedly lower than that for Rab5a.