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. 2017 Aug 10;7:7841. doi: 10.1038/s41598-017-08334-x

Figure 6.

Figure 6

Ang II induces the Smad activation in LX-2 cells. (A) Serum-starved LX-2 cells were incubated with Ang II (10−7 M) for 0, 0.25, 0.5, 1, 2 and 4 h. Whole cell lysates were subjected to immunoblotting analysis using phosphorylated Smad2/3 antibodies, and total protein levels of Smad2/3 were used as internal controls. Cytoplasmic and nuclear fractions were prepared and analyzed by Western blotting to detect Smad2/3 and Smad4. The β-actin and lamin B1 protein levels were used as internal controls, respectively, for cytoplasmic extracts (CE) and nuclear extracts (NE). (B–D) The protein levels of phosphorylated Smad2/3 in whole cell extracts, and Smad2/3 and Smad4 in cytoplasmic and nuclear extracts were converted to arbitrary densitometric units, normalized by the value of the corresponding loading control and expressed relative to the phosphorylation ratio or the protein level as n-fold over unstimulated cells (defined as 1-fold). (E) The localization of phosphorylated Smad2/3 was examined by immunofluorescence staining following treatment of LX-2 cells with Ang II (10−7 M) for 0.5 h. The fluorescence images were observed and captured by a confocal laser scanning microscope. Scale bar, 20 μm. (F) Serum-starved LX-2 cells were incubated with Ang II (10−7 M) for 0.5 h. Nuclear proteins were extracted and the binding activity of Smad3/4 with the consensus binding sequences was analyzed by EMSA, as described in Materials and Methods. The binding mixtures were separated on a 6% nondenaturing polyacrylamide and the DNA-protein complexes were visualized with an ECL assay Kit. (G) Serum-starved LX-2 were transfected with either Smad binding element-luciferase reporter plasmid (pSBE4-luc) or pBV-Luc reporter construct, and then stimulated by Ang II (10−7 M) for 0.5 h. The Renilla luciferase reporter plasmid was used as the internal control of transfection efficiency. The firefly or Renilla luciferase activity in total cell extracts was determined using the Dual-Luciferase Reporter Assay System. Experiments were repeated 3 times with similar results, and all data are presented as mean ± SD. # P < 0.05 versus unstimulated control cells; * P < 0.05 versus pSBE4-luc-transfected cells without Ang II stimulation.