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1.
Figure 4

Figure 4. Three major isoforms of ΔNp63 can induce expression of K5 in cell culture.. From: An Active Role of the ΔN Isoform of p63 in Regulating Basal Keratin Genes K5 and K14 and Directing Epidermal Cell Fate.

Ptk2 cells were transfected with plasmids encoding different HA-epitope tagged isoforms of ΔNp63 as indicated. Cells were stained with antibodies detecting HA (red) and K5 (green). Nuclei are stained with DAPI (blue). 40× magnification is shown.

Rose-Anne Romano, et al. PLoS ONE. 2009;4(5):e5623.
2.
Figure 6

Figure 6. Ectopic Expression of ΔNp63β can partially rescue the p63 null phenotype.. From: An Active Role of the ΔN Isoform of p63 in Regulating Basal Keratin Genes K5 and K14 and Directing Epidermal Cell Fate.

Top left panel shows H&E staining of p63−/− and p63−/−,ΔNp63β rescued animals. Scale bar: 50 µm. Remaining panels illustrate immunofluorescence staining using various antibodies as indicated (in green). White hashed line demarcates the dermal epidermal boundary. Scale bar is 25 µm for immunofluorescence images.

Rose-Anne Romano, et al. PLoS ONE. 2009;4(5):e5623.
3.
Figure 1

Figure 1. Generation of TAp63-specific antibody.. From: An Active Role of the ΔN Isoform of p63 in Regulating Basal Keratin Genes K5 and K14 and Directing Epidermal Cell Fate.

A) Specificity of the TAp63 antibody. Western blot analysis of whole cell extracts transfected with various HA-epitope tagged isoforms of p63 show reactivity of the TAp63 antibody with cells transfected with HA-TAp63α, TAp63β, and TAp63γ only. B) Immunofluorescence of cells transfected with HA-TAp63γ demonstrate the ability of the TAp63 antibody to detect TAp63 in cell culture in vivo. DAPI nuclear staining is shown in blue, HA epitope tag is shown in red, TAp63 specific staining is shown in green and a merge is shown in the lower panel. C) TAp63 expression in oocytes in sections of mouse ovary. TAp63 expression is shown in red and DAPI is shown in blue.

Rose-Anne Romano, et al. PLoS ONE. 2009;4(5):e5623.
4.
Figure 2

Figure 2. ΔNp63 is expressed during early mouse embryonic skin development.. From: An Active Role of the ΔN Isoform of p63 in Regulating Basal Keratin Genes K5 and K14 and Directing Epidermal Cell Fate.

Isoform specific expression of p63 was analyzed during various stages of skin development using three antibodies recognizing different p63 variants. Far left panel (panel 1) are H&E staining of skin using paraffin embedded whole embryos. Panel 2 demonstrates expression of ΔNp63 (green) using the RR-14 antibody. Panel 3 shows expression of p63α (green) using the H-129 antibody. K5 and K14 expression is also shown in green (panels 3 and 4 respectively). Far right panel reveals that TAp63 expression is absent during the developmental windows examined. Scale bar: 50 µm.

Rose-Anne Romano, et al. PLoS ONE. 2009;4(5):e5623.
5.
Figure 5

Figure 5. ΔNp63 can induce de novo expression of K5 and K14 in single-layered lung epithelia.. From: An Active Role of the ΔN Isoform of p63 in Regulating Basal Keratin Genes K5 and K14 and Directing Epidermal Cell Fate.

A) Schematic depicting the mating scheme used to generate transgenic animals expressing the HA-ΔNp63 transgene in single-layered lung epithelium using the surfactant protein C (SPC) promoter in a tetracycline inducible fashion. B) Gross morphology of E18.5 lung tissue sections stained with H&E from control animals and bi-transgenic animals (ΔNp63αBG and ΔNp63βBG). Bi-transgenic animals exhibit squamous metaplasia. Scale bar: 50 µm. C) Lung tissue sections from E18.5 stained with antibodies detecting HA epitope tag (green) and K5 or K14 (red) reveal expression of the transgene in bi-transgenic animals. Dapi staining is shown in blue. Tissues from bi-transgenic animals demonstrate induction of K5 and K14 expression (red staining) as compared to control littermates.

Rose-Anne Romano, et al. PLoS ONE. 2009;4(5):e5623.
6.
Figure 3

Figure 3. Binding of ΔNp63α to Hs II region of the K5 gene.. From: An Active Role of the ΔN Isoform of p63 in Regulating Basal Keratin Genes K5 and K14 and Directing Epidermal Cell Fate.

A) EMSA reveals binding of ΔNp63α in nuclear extracts of HaCaT cells using a radiolabeled probe corresponding to Hs II of the human K5 gene. Competition assays show that the KSC oligonucleotide, which contains a previously identified ΔNp63α binding site within the K14 gene, competes with binding of Hs II probe (lanes 2–3). A p53 consensus (lanes 4–5) and a WT oligonucleotide (lane 6–7) also compete for binding while a MT oligonucleotide did not affect Hs II binding (lanes 8–9). The highest complex (asterix) is supershifted (arrow) with the addition of antibodies raised against various domains of ΔNp63 whereas anti-TA antibodies have no effect (lanes 10–13). The lower complexes are likely to be non-specific. B) In vivo occupancy of ΔNp63α to the mouse and human K5 gene. ChIP was performed on HaCaT or primary mouse keratinocytes using two separate antibodies recognizing ΔNp63α as well as a nonspecific IgG as indicated (left panel). Input represents PCR amplification of 1% of the genomic DNA. Primers corresponding to a region of the GAPDH gene serves as a negative control. Right panel shows results obtained from real-time PCR experiments.

Rose-Anne Romano, et al. PLoS ONE. 2009;4(5):e5623.

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