Oxidative Medicine and Cellular Longevity / 2016 / Article / Fig 5

Research Article

Plant Natural Product Formononetin Protects Rat Cardiomyocyte H9c2 Cells against Oxygen Glucose Deprivation and Reoxygenation via Inhibiting ROS Formation and Promoting GSK-3β Phosphorylation

Figure 5

Effects of formononetin on the formation of ANT-Cyp-D complex and the binding of phospho-GSK-3β (Ser9) to ANT. (a) Immunoprecipitation with antibody against ANT. After the treatment with formononetin under OGD and reoxygenation condition, H9c2 cells were lysed for immunoprecipitation (IP) with antibody against ANT. The pull-down materials were analyzed by immunoblotting (IB) with antibodies against ANT and Cyp-D. (b) Immunoprecipitation with antibody against phospho-GSK-3β (Ser9). After the treatment with formononetin under OGD and reoxygenation condition, H9c2 cells were lysed for immunoprecipitation (IP) with antibody against phospho-GSK-3β (Ser9). The pull-down materials were analyzed by immunoblotting (IB) with antibodies against ANT and phospho-GSK-3β (Ser9). (c) Immunofluorescence imaging of the intracellular phospho-GSK-3β (Ser9). After the treatment with formononetin under OGD and reoxygenation condition, H9c2 cells were probed with antibody against phospho-GSK-3β (Ser9) and mitochondrial membrane protein Tom20 and subsequently detected by fluorophore-labelled secondary antibodies, phospho-GSK-3β (Ser9) (RED), and Tom20 (GREEN), respectively. The cell nuclei were indicated by DAPI staining. The images were captured under a fluorescence microscope (Carl-Zeiss, Jena, Germany). Scale bar, 10 μm.
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