Krüppel-Like Factor 5 Mediates Proinflammatory Cytokine Expression in Lipopolysaccharide-Induced Acute Lung Injury through Upregulation of Nuclear Factor-κB Phosphorylation In Vitro and In Vivo
Figure 5
Krüppel-like factor 5 (KLF5) interacts with nuclear factor-kappaB subunit in the nuclei of human bronchial epithelial cells. Cells from the HBEC line were treated with lipopolysaccharide (LPS; 5 μg/mL) for 1 h, with or without N-acetylcysteine (NAC) pretreatment (10 mM). The nuclear protein extracts were immunoprecipitated with KLF5 or anti-immunoglobulin G (IgG) antibodies (Abs) and then analyzed using western blotting and Abs against (a) KLF5 (molecular weight [MW]: 50 kDa), (b) p65 (MW: 65 kDa), (c) phospho-p65 (Ser276; MW: 65 kDa), and (d) phospho-p65 (Ser536; MW: 65 kDa). Nuclear protein extracts were immunoprecipitated with Abs against phospho-p65 (Ser276) or anti-IgG and then analyzed using western blotting and Abs against (e) phospho-p65 (Ser276) and (f) KLF5 to confirm the interaction between KLF5 and phospho-p65 (Ser276). The target protein was marked along with a nonspecific binding site. LPS-induced KLF5 coprecipitates with KLF5 (a), p65 (b), and phospho-p65 (Ser276) (c) but not with phospho-p65 (Ser536) (d). The results of immunoprecipitation with a phospho-p65 (Ser276) Ab and immunoblotting for phospho-p65 (Ser276) (e) and KLF5 (f) showed that phospho-p65 (Ser276) interacted with KLF5 proteins in cells from the HBEC line.
