The Interplay between Cyclic AMP, MAPK, and NF-κB Pathways in Response to Proinflammatory Signals in Microglia

Figure 4

TNF-α reduced levels of catalytic pPKAcatα^Thr197 and altered its subcellular localization while increasing cytosolic levels of activated pERK in EOC2 microglia. Compared to the levels of pPKAcatα^Thr197 (red) in resting EOC2 microglia (a–c), TNF-α stimulation (d–f) produced a significant reduction in pPKAcatα^Thr197 immunoreactivity as well as altered its subcellular localization from a diffuse to particulate cytosolic appearance at 3 h. Western blot analysis of the total cell lysates of untreated and TNF-α stimulated EOC2 microglia showed no change in total PKAcatα (g-h) and confirmed quantitatively the significant reduction in pPKAcatα^Thr197 within 3 h of stimulation, which remained depressed through 24 h (i-j). Quantification of pPKAcatα^Thr197 levels using immunoblot densitometry values (arbitrary units) normalized to β-actin (j). Conversely, pERK immunoreactivity (green), which was almost undetectable in resting microglia (k–m), was significantly increased within 3 h of stimulation with TNF-α (n–p). Western blot analysis confirmed the upregulation of pERK at 3 h post-TNF-α activation, though by 24 h these levels had returned to near normal values (q-r). Quantification of ERK levels by densitometry analysis (arbitrary units) normalized to β-actin (r). The results shown are the averages from three independent culture plate replicates for each treatment condition; the bands from all three (g) or two (o) of the replicates are presented on the blot images. Scale bars = 20 μm. Errors are given as SEMs. Statistical significance relative to naive controls is indicated at and versus 24 h post-TNF-α stimulation at or .