Figure 5 | Scientific Reports

Figure 5

From: Recombinant Mtb9.8 of Mycobacterium bovis stimulates TNF-α and IL-1β secretion by RAW264.7 macrophages through activation of NF-κB pathway via TLR2

Figure 5

rMtb9.8 induces the phosphorylation of NF-κB p65 at Ser536 and nuclear translocation in mouse macrophages. Analysis of the nuclear translocation of NF-κB in RAW264.7 cells incubated with rMtb9.8 (5 µg/ml) for 0, 10 and 30 min by ImageStream 100 is shown. A specific antibody against phospho-NF-κB was used to detect NF-κB phosphorylated at Ser536. Live cells were distinguished from dead cells by the presence of a single, well-formed DAPI-stained nucleus (live cells), after which we gated live cells and NF-κB-containing cells. Nuclear translocation was defined by the colocalization of NF-κB (green) with the nuclear stain DAPI (red); colocalization is indicated by the presence of a bright yellow nucleus in the overlaid images of live cells and focused NF-κB-containing cells. Top: From left to right, the images show cells that exhibit low to high nuclear translocation at time intervals of 0, 15 and 30 min. Bottom: From left to right, the images are as follows: bright field, DAPI stained, phospho-p65-stained and a merged image of DAPI and phospho-p65-stained cells. The data are representative of three independent experiments; 30,000–50,000 live focused cells were recorded in each experiment.

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