Figure 3.

Pull-down assay. (A) Results of GST pull-down assay to detect the interaction of GST-UreA and GST-UreB with Hsp60. (B) The interaction of Hsp60 with UreA-GST or UreB-GST was verified individually. All pull-down assays were performed as follows: 20 μl of glutathione Sepharose 4B beads saturated with GST-fused proteins were incubated with 0.4 ml of Hsp60 (1 mg/ml dissolved in 50 mM Tris-HCl, pH 8.0) at 4°C for 2 h. The pellets were washed four times and resuspended in SDS-PAGE sample buffer for Western blotting (W) and SDS-PAGE (S) analysis. Hsp60 co-incubated with GST-tag immobilized beads and empty beads were used as negative controls. Purified Hsp60 was used as a positive control. Anti-His antibody was used to detect the bound Hsp60 and horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG 158 was used to detect the primary antibody. All experiments were repeated at least three times.