Figure 3

IκKα/β signaling in the impaired monocyte. (A) Primary monocytes were cultured in either media only (naïve) or 10 ng/mL of LPS for 16 h, washed and resuspended in fresh media and challenged with 100 ng/mL of LPS for 60 min. Total protein was analyzed by Western blots for IκBα and normalized to vinculin (n = 5). (B, C) Primary monocytes were cultured for 16 h in media only (naïve) or 10 ng/mL of LPS (impaired) and total protein concentrations of IκK-α & IκK-β were determined by normalizing to β-actin (n = 8 for each). (D) After 16 h of incubation in naïve or impaired conditions, IκK-α & IκK-β mRNA expression was measured by qRT-PCR normalizing to 18S (n = 7). (E, F) Monocytes were cultured in naïve or impaired conditions for 16 h, washed and then challenged with 100 ng/mL of LPS. Phosphorylation of IκKα/β was determined by Western blot and normalized to β-actin (n = 6). Original uncropped gels are available in Supplemental Material. Data is shown as mean ± SE. *p < 0.05, Wilcoxon signed-rank test.