Figure 5
AMWAP is taken up by microglia and inhibits LPS-mediated NFκB activation. (A-C) BV-2 microglia were incubated with fluorescently labeled recombinant AMWAP (AMWAP-Fluo EX, 10 μg/ml) for 6 and 24 h. Microglia gradually incorporated AMWAP-Fluo EX into their cytosol exhibiting a perinuclear localization as shown by anti-Iba1 antibody co-staining. (D-G) AMWAP-Fluo EX pretreatment (for 1 to 24 h) time-dependently inhibited TLR4-mediated nuclear translocation of NFκB p65 after stimulation with 50 ng/ml LPS for 1 h as shown by immunocytochemistry and quantification of nuclear vs. cytosolic fluorescence intensities. PBS served as vehicle control. Data show mean ± SD (n = 9/group) with *P < 0.05, **P < 0.01, ***P < 0.001 for AMWAP-treated vs. control cells. (H) Control and AMWAP-treated BV-2 microglia were stimulated with 50 ng/ml LPS for 1 h and NFκB p65 protein amount was determined in cytosolic (c) and nuclear (n) fractions using Western blot. AMWAP-treated cells exhibited diminished LPS-triggered NFκB p65 translocation to the nucleus compared to vehicle control. (I) Control and AMWAP-treated BV-2 microglia were stimulated with 50 ng/ml LPS for 1 h and the NFκB p65 phosphorylation status (pNFκB p65) was determined in total cell lysates using Western blot. Actin served as loading control. Scale bar = 20 μm.