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. Author manuscript; available in PMC: 2010 Oct 2.
Published in final edited form as: DNA Repair (Amst). 2009 Aug 4;8(10):1215–1224. doi: 10.1016/j.dnarep.2009.07.003

Fig. 2.

Fig. 2

Interaction of MDM2 and p53 with RPS3 by protein pull-down assays. (A) Purified His-MDM2 immobilized on to NiNTA beads were mixed with GST or GST-RPS3 contained in binding buffer and incubated for 1 h at room temperature. Beads were then collected, washed and protein complexes eluted into Laemmli buffer. After resolving the protein complexes on SDS-PAGE, the interaction of RPS3 with MDM2 was analyzed by immunoblotting by probing the membrane with GST and MDM2 antibodies. (B) Reaction mixtures containing His-p53 bound to NiNTA beads and purified GST or GST-RPS3 proteins were processed as in (A) and the interaction of RPS3 with p53 was demonstrated by using an antibody to GST. (C) GST pull-down was performed by incubating HEK 293 whole cell lysate with GST (lane 2), GST-RPS3 (lane 3) or GST-RPS3-ΔKH (lane 4) immobilized onto agarose beads. After elution and SDS-PAGE, proteins were observed by Coomassie staining (left panel) and identified by immunoblotting with p53 and MDM2 antibodies (right panel). The whole cell lysate used for the pull-down (lane 1) is shown as a control for total protein.