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Fig. 6 | Retrovirology

Fig. 6

From: Identification of novel HIV-1 dependency factors in primary CCR4+CCR6+Th17 cells via a genome-wide transcriptional approach

Fig. 6

NF-κB nuclear translocation and DNA-binding activity in CCR4+CCR6+ Th17 versus CXCR3+CCR6 Th1 cells. a, b Matched Th17 and Th1 subsets were isolated and stimulated via CD3/CD28 for 3 days as described in Fig. 1. Cells were seeded on poly-l-lysine coated slides, and intracellular staining was performed with rabbit anti-human NF-kB followed by goat anti-rabbit AlexaFluor 488 Abs. Slides were mounted with the ProLong Gold Antifade reagent containing the nuclear dye DAPI. Slides were then observed by confocal microscopy. a NF-κB expression was observed under a 100× oil-immersion objective (NA = 1.46) in a spinning-disc confocal mode system. Shown are maximum intensity z projection of z stack of Th17 and Th1 cells from one donor representative of observations made with two different donors. b The relative expression of intra-nuclear NF-κB was quantified in cells from two different donors using the Image J software based on observations by epifluorescence with a 40x Oil immersion objective (NA = 1.40). Horizontal red lines indicate median values. Unpaired t test p values are indicated in the figure. c Memory CCR6+ and CCR6 CD4+ T-cell subsets were enriched by MACS and sorted by FACS as previously reported [104]. Cells were stimulated via CD3/CD28, as in Fig. 1, and nuclear fractions were extracted. Shown is the ELISA quantification of NF-κB-p65 DNA-binding activity in nuclear extracts from matched CCR6+ and CCR6 subsets isolated from three different HIV-uninfected donors (mean ± SD of triplicate wells). Student t test p values are indicated in the figures

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