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Figure 6 | Retrovirology

Figure 6

From: Caspase-3-mediated cleavage of p65/RelA results in a carboxy-terminal fragment that inhibits IκBα and enhances HIV-1 replication in human T lymphocytes

Figure 6

Analysis of DNA-binding and transcriptional activity of ΔNH 2 p65. (a) Proteins p65wt-tag, p65 D94E;D97E-tag, ΔNH2p65-tag and NF-κB1/p50 were expressed in vitro by using a wheat germ-based transcription-translation system. Protein expression was confirmed by immunoblotting with specific antibodies against the carboxy-terminus of p65/RelA and NF-κB1/p50. (b) Three micrograms of in vitro translated p65wt-tag, p65 D94E;D97E-tag and ΔNH2p65-tag proteins, as well as, NF-κB1/p50 were analyzed separately (homodimers) or together (heterodimers) by EMSA using a [α-32P]-dCTP-labeled double-stranded synthetic wild-type HIV enhancer oligonucleotide containing two -κB consensus motifs. The nucleoprotein-oligonucleotide complexes were analyzed by electrophoresis on non-denaturing polyacrylamide gel. (c) Jurkat cells were transiently transfected with pCMV-p65wt-tag, pCMV-p65 D94E;D97E-tag or pCMV-ΔNH2p65-tag along with the plasmid pκB-conA-LUC, which contains the luciferase (LUC) gene under the control of three consensus sites for NF-κB. After 18 hours of incubation in the absence of activation, protein extracts were analyzed for relative luciferase units (RLUs) expression. Internal control of transfection was carried out by co-transfection with pSV-β-Galactosidase vector and protein concentration was also measured to normalize the data. Data correspond to the mean of three different experiments and lines on the top of the bars represent the standard deviation.

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