Fig. 5

Inhibition of MyD88-dependent TLR signaling abrogates N-cdh-stimulated TNF-α release and NF-κB translocation. Microglia were pre-treated with either 200 μM of control peptide (CTLpep) or inhibitory MyD88 peptide (MyD88pep) for 30 min, followed by a 2-h exposure to 75 nM soluble N-cdh (a–c). Supernatants were collected and fixed cells were immunostained for NF-κB (rabbit polyclonal anti-p65, 1:1000; Alexa Fluor 594-conjugated goat anti-rabbit IgG secondary antibody, 1:1000) and counterstained with DAPI, while cell culture supernatants were subsequently analyzed for TNF-α protein concentration via ELISA (c). Data shown is representative and from one of two separate experiments each performed in triplicate. As shown, MyD88 inhibition led to a reduction in N-cdh-stimulated NF-κB translocation. Representative immunostaining is shown in (a) and quantification of nuclear signal intensity change is shown in (b) (n = 60 control and 84 MyD88 inhibitor-treated cells). The difference between nuclear signal intensity in vehicle and N-cdh is significant at *p < 0.05. The scale bar again represents 20 μm. In addition, as shown in (c), inhibition of MyD88 homodimerization resulted in a significant decrease in N-cdh-stimulated TNF-α release from microglia in comparison to control conditions (*p < 0.05). Arrows in CTLpep indicate nuclear NF-κB, which is diminished with the inhibitory MyD88 peptide