Figure 6
Rabbit reticulocyte lysate (RRL) produced p53 and IκBα proteins interact in vitro. A. Wild-type IκBα and mutant proteins were synthesized in rabbit reticulocyte lysates (lane 1, RRL no RNA; lane 2, ΔA2; lane 3, ΔA3; lane 4, ΔA4; lane 5, ΔA5; and lane 6, wild-type) and precipitated with a rabbit polyclonal antiserum against the C-terminus of IκBα using protein A/G sephadex beads either in the absence (p53-) or in the presence (p53+) of RRL synthesized, [35S]-labeled, p53. Equal volumes of RRL were used in all cases. For p53- reactions, RRL programmed with empty vector was incubated with [35S]-labeled methionine. A control reticulocyte lysate without IκBα protein template (lane 1) was included in these experiments. ΔA2 (lane 2), ΔA3 (lane 3), ΔA4 (lane 4) ΔA5 (lane 5) and wild-type (lane 6) IκBα proteins were co-incubated with p53, precipitated and analzyed by SDS-PAGE followed by radiography. B. Wild-type p53 protein translated in RRL (lane 1) produced both putative full-length p53 protein (denoted p53, mobility approximately 55 kD) and also at least two less-than-full length translation products (denoted * and **; lanes 1–3). Putative full-length and less-than full-length p53 translation products were precipitated by p53-specific monoclonal antibodies and protein A/G sephadex beads (lane 2, Ab1 recognizing the carboxyl terminus of p53; lane 3, Ab2 recognizing the amino terminus of p53). Less-than full-length proteins were more readily precipitated by Ab1 than Ab2. Control precipitation with IgG2A immunoglobulin (lane 4) or protein G/A beads (lane 5) did not precipitate p53. C. IκBα proteins were precipitated in similar quantities by IκBα antiserum. A shorter exposure of the gel than in panel A is shown. D. Densitometry of p53 protein precipitated in association with various IκBα alleles. Data shows significant precipitation of p53 with ΔA4, ΔA5 and wild-type, expressed as a ratio of label precipitating with IκBα allele to beads alone. Data was calculated by first normalizing to the background in each lane followed by calculating the ratio of label precipitating with IκBα to a corresponding area in the beads alone lane.