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. 1999 Feb;19(2):1092–1100. doi: 10.1128/mcb.19.2.1092

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Copyright © 1999, American Society for Microbiology

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FIG. 5 — Interactions of p53_Pro and p53_Arg with transcription factors. (A) GST fusion protein pull-down assay with GST-p53_Pro and GST-p53_Arg bound to glutathione-agarose. In vitro-translated (ivt) ³⁵S-labeled p53_Pro, p53_Arg, TAFII70, or TBP proteins were incubated with the fusion protein-agarose at either 0°C or room temperature (rt). The bound proteins were analyzed by SDS-PAGE and autoradiography. No differences in the abilities of p53_Pro and p53_Arg to form either hetero- or homodimers or in their abilities to bind to TBP were apparent. However, TAFII70 is bound significantly only at room temperature, and p53_Pro binds more strongly than p53_Arg to TAFII70. (B) The GST-fusion protein pull-down assay repeated with in vitro-translated ³⁵S-labeled TAFII32 and TAFII250 plus p53_Pro (P) and p53_Arg (R) for comparison. TAFII32 binds more strongly at room temperature and binds more strongly to p53_Pro than to p53_Arg. TAFII250 binds to p53_Pro and p53_Arg with similar affinities but binds more strongly at room temperature. (C) Phosphorimager analysis of at least five GST fusion protein pull-down assays. The fold increase in binding at room temperature over binding at 0°C is shown. p53_Pro binds approximately twice as strongly as p53_Arg to TAFII32 and TAFII70. GST alone was also used as a negative control; in all cases, the percentage of loaded protein retained was less than 1%.

FIG. 5 — Interactions of p53_Pro and p53_Arg with transcription factors. (A) GST fusion protein pull-down assay with GST-p53_Pro and GST-p53_Arg bound to glutathione-agarose. In vitro-translated (ivt) ³⁵S-labeled p53_Pro, p53_Arg, TAFII70, or TBP proteins were incubated with the fusion protein-agarose at either 0°C or room temperature (rt). The bound proteins were analyzed by SDS-PAGE and autoradiography. No differences in the abilities of p53_Pro and p53_Arg to form either hetero- or homodimers or in their abilities to bind to TBP were apparent. However, TAFII70 is bound significantly only at room temperature, and p53_Pro binds more strongly than p53_Arg to TAFII70. (B) The GST-fusion protein pull-down assay repeated with in vitro-translated ³⁵S-labeled TAFII32 and TAFII250 plus p53_Pro (P) and p53_Arg (R) for comparison. TAFII32 binds more strongly at room temperature and binds more strongly to p53_Pro than to p53_Arg. TAFII250 binds to p53_Pro and p53_Arg with similar affinities but binds more strongly at room temperature. (C) Phosphorimager analysis of at least five GST fusion protein pull-down assays. The fold increase in binding at room temperature over binding at 0°C is shown. p53_Pro binds approximately twice as strongly as p53_Arg to TAFII32 and TAFII70. GST alone was also used as a negative control; in all cases, the percentage of loaded protein retained was less than 1%.