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Figure 2 | Retrovirology

Figure 2

From: High level expression of the anti-retroviral protein APOBEC3G is induced by influenza A virus but does not confer antiviral activity

Figure 2

IFNβ-induced transcription of human APOBEC3G. (A and B) A549 cells stably overexpressing the dominant negative mutants IKK2KD or MKK6Ala. These mutant kinases were cloned in the retroviral pEGZ-vector. In this vector GFP is expressed by an internal ribosomal entry site (IRES) from the same mRNA as the IKK2KD and MKK6Ala transgenes, allowing transgene expression to be monitored by FACS-analysis (data not shown). MKK6Ala and IKK2KD overexpressing cells were infected for 10 hours with the influenza A virus strain PR8 (MOI = 5). Following infection, RNA was isolated with the RNeasy mini kit (Qiagen), reverse transcribed as described above, and cDNA was subjected to qRT-PCR. (C) A549 cells were pre-treated for 30 minutes with the NF-κB specific inhibitor BAY 11–7085 (40 μM) before infection with PR8 (MOI = 5) for 10 hours. RNA was subjected to qRT-PCR. The mRNA levels of human APOBEC3G (A and C) or IFNβ (B) were assessed by qRT-PCR. (D) A549 cells were stimulated with IFNβ (100 U/ml) (Invitrogen) for the time points indicated. The mRNA levels of human APOBEC3F and human APOBEC3G were determined by means of qRT-PCR. Induction of gene transcription was calculated as n-fold of untreated cells, which was arbitrarily set as 1, as described by Livak and Schmittgen. (E) A549 cells were stimulated as in (D), and cell lysates were subjected to Western blots using the hA3G specific antibody ApoC17 (NIH)

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