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Figure 3 | BMC Immunology

Figure 3

From: Modulation of p53 activity by IκBα: Evidence suggesting a common phylogeny between NF-κB and p53 transcription factors

Figure 3

P53 and IκBα proteins co-precipitate in vitro. A: Purified bacterially produced IκBα protein co-precipitated specifically with p53. Purified IκBα protein was specifically labeled with [γ-32P]ATP using p90rsk (lane 5). [32P]-labeled IκBα was incubated with purified p53 (GST-p53, lanes 1–3) or a control GST fusion protein (GST-c-Jun, lane 4). P53 was precipitated either by a glutathione binding tag on GST-p53 and glutathione Sepharose beads (Seph-GSH, lane 1) or a p53 specific antibody (Ab2, lane 2) and protein A/G Sephadex beads. IκBα protein (position indicated) was not precipitated by p53 specific Ab5 (lane 3) that does not recognize bacterially synthesized p53 protein, or by incubation with GST-c-Jun and precipitation with glutathione Sepharose beads (lane 4). Proteins were separated by PAGE and detected by autoradiography of [32P]-labeled protein. Electrophoresis of the input IκBα used in this experiment is also shown in lane 5. B: Relatively less ΔN1 protein than ΔC1 IκBα protein co-precipitated with GST-p53 from COS cell lysates. After expression of IκBα in COS cells, whole cell lysates were incubated with bacterially produced purified p53 (GST-p53). P53/ IκBα complexes were then precipitated with glutathione Sepharose beads and analyzed by PAGE/Western blotting. In the left panels, ΔN1 and ΔA2 proteins were detected using a rabbit polyclonal antisera directed at the C-terminus of IκBα, while in the right panels, ΔA2 and ΔC1 proteins were detected using a rabbit polyclonal antisera directed at the N-terminus of IκBα. C: Structures of wild-type IκBα and mutant constructs used in these studies.

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