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Fig. 2 | Journal of Neuroinflammation

Fig. 2

From: Matrix metalloproteinase activity stimulates N-cadherin shedding and the soluble N-cadherin ectodomain promotes classical microglial activation

Fig. 2

Soluble N-cdh acts on primary microglial cells to stimulate nuclear translocation of NF-κB and increased levels of Iba-1 in lysates. Microglia were immunofluorescently labeled for the p65 subunit of NF-κB (rabbit polyclonal anti-p65, 1:1000; Alexa Fluor 594-conjugated goat anti-rabbit IgG secondary antibody, 1:1000) and counterstained with DAPI, following a 2-h exposure to either vehicle (PBS) or 75 nM of soluble N-cdh. In comparison to control conditions, incubation of microglia with soluble N-cdh induced a robust increase in nuclear NF-κB p65 immunoreactivity, consistent with classical microglial activation. Representative images are shown in (a) and quantification in (b) (p < 0.01). The scale bar in (a) (white) represents 20 μm. Western blot analysis (c) shows that Iba-1 protein levels were also increased in microglial lysates (12 μg protein/lane; anti-Iba1 rabbit polyclonal, 1/1000; HRP-goat anti-rabbit IgG, 1/3000) following an 18-h exposure of microglia to 75 nM of N-cdh. Membranes were subsequently reprobed for GAPDH as a loading control (anti-GAPDH mouse monoclonal IgG1, 1/20,000; HRP-goat anti-mouse IgG, 1/2000). Data in (d) show results of densitometric analysis comparing changes in Iba-1 signal relative to GAPDH in control or N-cdh-stimulated cell lysates. The difference between the control and N-cdh-stimulated Iba-1/GAPDH density ratio is significant at p < 0.05. Arrows indicate increased nuclear NF-κB immunoreactivity

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