Figure 3. HGFL treatment impacts IκB protein levels and inhibits NF-κB activation in MH-S cells following LPS stimulation.

Cell lysates from the murine alveolar macrophage cell line, MH-S, were obtained after overnight pretreatment in the presence or absence of HGFL (400ng/ml) followed by 4 hours of treatment with or without LPS. (A) Western analysis was performed on the MH-S cell lysates to examine IκBα and IκBβ protein expression levels. Actin served as a loading control. Each lane represents an independent sample and is representative of at least two independent experiments. (B) Electrophoretic mobility shift assays on whole cell extracts from MH-S cells were performed to examine NF-κB DNA binding activity in response to LPS alone or LPS plus HGFL pretreatment. The specific NF-κB DNA complex is denoted by the arrow with free probe also indicated. Competition and supershift analyses for NF-κB-DNA binding proteins were also performed. The protein-DNA complexes were competed with either 100-fold excess of cold probe or with antibodies directed against the p50 and p65 subunits of NF-κB. (C) MH-S cells were transfected with an NF-κB reporter plasmid along with a control plasmid. After transfection, the cells were pretreated in the presence or absence of HGFL overnight and the next day stimulated with or without LPS. Relative NF-κB activity for each sample is depicted. Data are expressed as the mean ± standard error.