Figure 6.
Figure 6. The domain of p65 between amino acids 305 to 521 is important for Tax-mediated p53 inhibition. (A) A schematic diagram of the p65 constructs used in transfection studies in p65–/– cells is shown. (B) p65–/– cells were transiently cotransfected with use of Effectene reagent (Qiagen) with the PG13-Luc reporter construct and control (con), wild-type p65 (WT, 0.1 μg), or p65 deletion constructs 521 or 305 in the absence (□) or presence (▪) of Tax (0.3 μg). A representative experiment is shown in which the p53 activity in the absence of Tax is set at 100%. The RLTK-Luc construct was included in the transfections to equilibrate for transfection efficiency. (C) Protein expression levels of the p65 constructs were determined by Western blot analysis (p65 Ab2; Oncogene Research Product). (D) p65–/– cells were transfected (Lipofectamine plus reagent; Invitrogen) with WT, 521, or 305 p65 constructs or control DNA in the presence (+) or absence (–) of Tax. Nuclear extracts were prepared, and p65 complexes were isolated with use of anti-p65 antibody (Ab2; Oncogene Research Product). Immunocomplexes were separated on 8% Tris-gylcine gels and transferred to PVDF membranes, and the levels of associated p53 protein were visualized by chemiluminescence with an anti-p53 antibody (Ab1; Oncogene Research Product). (E) Western blot analysis was performed on 50 μg nuclear extract from transfected p65–/– cells to determine the levels of Tax and p53. (F) Wild-type (WT; 0.4 μg) or serine-to-alanine mutants (Ser15Ala, Ser37Ala, Ser392Ala, Ser15, 392Ala, or Ser15, 37Ala) of p53 were cotransfected in the absence (–) or presence (+) of Tax (2 μg) and the PG13-Luc reporter (1 μg) in p53–/–, MDM2–/– MEF cells. Luciferase activity was measured 24 hours after transfection. The graph indicates percentage of activity whereby the p53 activity for each construct was set to 100%. (G) Nuclear extracts were prepared 48 hours after transfection. Immunocomplexes were isolated from 0.5 mg nuclear extract with use of p53 (Ab6; Oncogene Research Product) antibody, separated on 10% Tris-glycine gels (Invitrogen), and transferred to PVDF membranes (Immobilon), and p65 was visualized with anti-p65 antisera (CT; Upstate). The lower panel indicates the amount of p53 immunoprecipitated from each extract (Ab7; Oncogene Research Product).

The domain of p65 between amino acids 305 to 521 is important for Tax-mediated p53 inhibition. (A) A schematic diagram of the p65 constructs used in transfection studies in p65–/– cells is shown. (B) p65–/– cells were transiently cotransfected with use of Effectene reagent (Qiagen) with the PG13-Luc reporter construct and control (con), wild-type p65 (WT, 0.1 μg), or p65 deletion constructs 521 or 305 in the absence (□) or presence (▪) of Tax (0.3 μg). A representative experiment is shown in which the p53 activity in the absence of Tax is set at 100%. The RLTK-Luc construct was included in the transfections to equilibrate for transfection efficiency. (C) Protein expression levels of the p65 constructs were determined by Western blot analysis (p65 Ab2; Oncogene Research Product). (D) p65–/– cells were transfected (Lipofectamine plus reagent; Invitrogen) with WT, 521, or 305 p65 constructs or control DNA in the presence (+) or absence (–) of Tax. Nuclear extracts were prepared, and p65 complexes were isolated with use of anti-p65 antibody (Ab2; Oncogene Research Product). Immunocomplexes were separated on 8% Tris-gylcine gels and transferred to PVDF membranes, and the levels of associated p53 protein were visualized by chemiluminescence with an anti-p53 antibody (Ab1; Oncogene Research Product). (E) Western blot analysis was performed on 50 μg nuclear extract from transfected p65–/– cells to determine the levels of Tax and p53. (F) Wild-type (WT; 0.4 μg) or serine-to-alanine mutants (Ser15Ala, Ser37Ala, Ser392Ala, Ser15, 392Ala, or Ser15, 37Ala) of p53 were cotransfected in the absence (–) or presence (+) of Tax (2 μg) and the PG13-Luc reporter (1 μg) in p53–/–, MDM2–/– MEF cells. Luciferase activity was measured 24 hours after transfection. The graph indicates percentage of activity whereby the p53 activity for each construct was set to 100%. (G) Nuclear extracts were prepared 48 hours after transfection. Immunocomplexes were isolated from 0.5 mg nuclear extract with use of p53 (Ab6; Oncogene Research Product) antibody, separated on 10% Tris-glycine gels (Invitrogen), and transferred to PVDF membranes (Immobilon), and p65 was visualized with anti-p65 antisera (CT; Upstate). The lower panel indicates the amount of p53 immunoprecipitated from each extract (Ab7; Oncogene Research Product).

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