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. 2005 Sep 15;19(18):2122–2137. doi: 10.1101/gad.1339905

Figure 7.

Figure 7.

Endogenous p53 isoforms are expressed at the protein level. (A) Transfection of p53 isoforms in H1299. A p53 variant can lead to p53 protein isoform expression. H1299 cells, devoid of p53 expression, were transfected with p53 cDNA variants cloned in mammalian expression vectors as indicated. Proteins were extracted 24 h after transfection, and analyzed by Western blot using either sheep polyclonal (sp) anti-human p53 or mouse monoclonal (DO-12) anti-human p53 (Vojtesek et al. 1995). (B) Endogenous p53 protein isoforms can be detected in human cell lines. Proteins from human cell lines expressing wild-type p53 (293) or mutant p53 (T47D, SW620, HT-29), or devoid of p53 expression (H1299, Saos-2) were extracted as described in Materials and Methods. Ten micrograms of protein extract were analyzed by Western blot. p53 expression was revealed using DO-12 mouse monoclonal antibody. (C) Anti-p53β antibody (KJC8) is specific for p53β and Δ133p53β. H1299 cells, devoid of p53 expression, were transfected with p53, p53β, Δ133p53β, or empty expression vectors. Protein extracts were analyzed by Western blot using either CM1 rabbit polyclonal anti-human p53 or peptide affinity-purified KJC8 rabbit polyclonal anti-p53β antibody. (D) U2OS cells express endogenous p53β and Δ133p53β protein isoforms. U2OS cells were transfected with 400 nM final concentration of small interference RNA oligonucleotides specific for p53 (Smartpool p53siRNA; Dharmacon) or control (Smartpool controlsiRNA; Dharmacon). Cells were harvested 96 h after transfection, and proteins were analyzed by Western blot using CM1 rabbit polyclonal anti-human p53 or KJC8 rabbit polyclonal anti-p53β. The anti-actin antibody was used to control loading and transfer efficiency. (E) p53β and Δ133p53β are not accumulated in MCF7 tumor cells in response to Actinomycin-D treatment. MCF7 cells expressing wild-type p53 were untreated or treated with 60 ng/mL of the DNA-damaging drug Actinomycin-D (Act-D), a potent p53 inducer. Proteins were extracted 6 h after treatment and analyzed (5 μg) by Western blot using either DO-1 mouse monoclonal anti-human p53 or peptide affinity-purified KJC8 rabbit polyclonal anti-p53β antibody. The anti-actin antibody was used to control loading and transfer efficiency.