Fig. 2

Human microglia constitutively and functionally express NK-1R. Panel a The hμglia immortalized human microglial cells are positive for Iba-1 (red) and constitutively express cell surface NK-1R (green) as determined by immunofluorescence microscopy. Nuclear staining is also shown in this representative image (DAPI: blue). Panel b hμglia cells were untreated (C) or exposed to bacterial flagellin (F: 10 or 25 ng/mL), Pam3Cys (P: 10 or 25 ng/mL), or LPS (1 or 5 ng/mL), in the presence or absence of SP (10 nM) for 18 h and protein expression of NK-1R and the housekeeping gene product β-actin was determined by immunoblot analysis (n = 4). The average relative expression of fNK-1R, determined by densitometric analysis and normalized to β-actin levels, is shown below the representative immunoblot. Panel c Primary human microglia were untreated (0) or exposed to LPS (5 ng/mL) for 18 h and protein expression of NK-1R and the housekeeping gene product β-actin were determined by immunoblot analysis (n = 2). Panel d Human microglia were untreated (0) or exposed to SP (10 nM) for 15, 30, 60, 90, and 120 min, and nuclear levels of NF-kB p65 (RelA) and the housekeeping gene product β-actin were determined by immunoblot analysis (n = 5). The average relative nuclear expression of RelA, determined by densitometric analysis and normalized to β-actin levels, is shown below the representative immunoblot. Data is expressed as the mean ± SEM and asterisks indicate a statistically significant difference from untreated cells (p < 0.05)