Figure 2

α ₁ -antitrypsin (A1AT)'s reduction of pro-inflammatory mediators is independent of MAPK and PKA activation. A. To detect the phosphorylation state of the MAPKs p38, p44/42 and JNK upon co-treatment with A1AT, primary microglial cells were incubated with 0.1 μg/ml lipopolysaccharide (LPS) and 2 and 4 mg/ml A1AT for 20 minutes, and cell lysates of the treated cells were subjected to western blotting. Antibodies against phospho-p38, phospho-p44/42 and phospho-JNK were used. GAPDH served as a loading control. One representative western blot out of three is shown. B. Band intensities of phosphorylated MAPK were measured by a densitometer and normalized with that of GAPDH. LPS-treated cells were set to 1. Means and standard deviations of the mean of three western blots are shown (*P value ≤ 0.05, **P value ≤ 0.01) C. To detect the effect of A1AT on the phosphorylation state of CREB, cells were pretreated with the PKA inhibitor H89 for 30 minutes and then treated with 4 mg/ml A1AT for 15 minutes or co-treated with 0.1 μg/ml LPS and 4 mg/ml A1AT for 15 minutes. Cell lysates were subjected to western blotting, and phospho-CREB was detected with a monoclonal antibody. Vinculin was used as a loading control. One representative western blot out of three is shown. D. To measure the impact on the release of the pro-inflammatory cytokine TNF-α, cells were pretreated with different concentrations of H89 and then treated with 0.1 μg/ml LPS and/or 2 mg/ml A1AT for 24 hours. TNF-α levels in cell culture supernatants of primary microglial cells were quantified with ELISA. Results were normalized to LPS-treated cells. Means and standard deviations of the mean of three independent experiments are shown.