Figure 3
Effects of LPS and LPS/A1AT-0 and LPS/A1AT-LA on Re/A/p65, NFKBIA/IκBα and NFKBIB/IκBβ expression and protein levels. Human neutrophils (5 × 106) were incubated for 5 h in either medium alone or medium containing LPS (20 ng/mL) in the absence or presence of A1AT-0 (1 mg/mL) or A1AT-LA (1 mg/mL). mRNA expression of Re/A/p65 (A), NFKBIA/IκBα (B) and NFKBIA/IκBβ (C) genes was analyzed as described in Materials and Methods. GAPDH and GUSB were used as housekeeping genes in qPCR experiments. Box plots represent data from 3 to 5 individual donors; n = number of replicates for each experiment. P value indicates significant differences compared with the values seen in LPS-activated cells. Subcellular fractions were analyzed by western blotting with anti-p65 (D), anti-NFKBIA/IκBα (E) and anti-NFKBIA/IκBβ (F) antibodies. The blots were reprobed with anti-β-actin and anti-histone H1 antibodies as loading controls for cytosolic and nuclear fractions, respectively. Each blot is representative of 3 independent donors.