Figure 5
Transient transfection of IκBα alleles specifically blocks p53 transcription in EBV-positive Akata cells. A. 200 ng of a p53-dependent reporter plasmid encoding 13 copies of the p53 response element driving a firefly luciferase (pG(13)Py/Luc) were cotransfected into 2 × 105 cells with 1 ng of an SV40 promoter driven Renilla luciferase (pRLSV40) and 200 ng of a CMV-promoter driven wild-type p53 cDNA construct (wt-p53, denoted below the x-axis) or a C-terminally deleted transcriptionally active p53 (ΔC-p53, denoted below the x-axis). The effect of 800 ng of CMV-promoter driven IκBα alleles or a control construct that did not contain DNA encoding IκBα (to which all data were normalized) were used to determine the effect of IκBα on wild-type p53-mediated transcription of pG(13)Py/Luc while 1600 ng of CMV-promoter driven IκBα alleles (or control) were used in experiments where the ΔC-p53 allele was used. Immunoblotting controls for wt-p53, ΔC-p53 and IκBα alleles. B: Western blot analysis of wt-p53 and ΔC-p53 in the presence of transfected IκBα alleles. Extracts were derived from Akata cells as described for Figure 5A. None indicates transfection of empty CMV vector containing no IκBα allele. C: Western blot analysis for IκBα in the presence of transfected wt-p53. A subset of extracts were analyzed both with C-term and N-term directed IκBα antibodies. Endogenous IκBα is detected by these methods. D: Western blot analysis of IκBα in the presence of ΔC-p53. ΔA3 was analyzed on a separate gel due to space limitations and showed a band intensity similar to that of ΔA4 and ΔA5 (data not shown).