Figure 5.

Ang II activates the NF-κB pathway in LX-2 cells. (A) Serum-starved LX-2 cells were treated with Ang II (10⁻⁷ M) for 0, 0.25, 0.5, 1, 2 and 4 h. Whole cell lysates were immunoblotted with antibodies against IκBα and its phosphorylated form. β-Actin was used as an internal control. Cytosolic and nuclear extracts were analyzed by immunoblotting for detection of NF-κB p65. The β-actin and lamin B1 protein levels served as internal controls, respectively, for cytoplasmic extracts (CE) and nuclear extracts (NE). (B) The protein levels of phosphorylated IκBα and IκBα in whole cell lysates, and (C) that of NF-κB p65 in cytoplasmic and nuclear extracts, were converted to arbitrary densitometric units, normalized by the value of the corresponding internal control and expressed according to the levels in unstimulated cells (defined as 1-fold). (D) Immunofluorescence staining was carried out with primary NF-κB p65 antibody followed by incubation of a FITC-labeled secondary antibody (green). The nuclei were stained with PI (red). In the merged micrographs, the yellow color indicates nuclear localization signal of NF-κB p65 subunit. Scale bar, 20 μm. (E) Serum-starved LX-2 cells were stimulated in the absence or presence of Ang II (10⁻⁷ M) for 0.5 h. Nuclear extracts were prepared and the binding activity of NF-κB with the consensus binding sequences was analyzed by EMSA, as described in Materials and Methods. The DNA-protein interaction was resolved on a 6% native polyacrylamide gel and the biotin-labeled DNA bands were visualized by an ECL assay Kit. (F) Serum-starved LX-2 cells were transfected with either firefly luciferase reporter of NF-κB (pNF-κB-luc) or pGL6-luc reporter construct followed by incubation with Ang II (10⁻⁷ M) for 0.5 h. The Renilla luciferase reporter was also co-transfected into the cells for normalizing transfection efficiency. The luciferase activity in the cellular extracts was evaluated by the Dual-Luciferase Reporter Assay System and normalized to the activity of Renilla. Experiments were repeated 3 times with similar results, and all data are presented as mean ± SD. ^# P < 0.05 versus unstimulated or pGL6-luc empty vector-transfected control cells; ^* P < 0.05 versus pNF-κB-luc-transfected cells without Ang II stimulation.