Journal: Basic research in cardiology
Article Title: ROS and endothelial nitric oxide synthase (eNOS)-dependent trafficking of angiotensin II type 2 receptor begets neuronal NOS in cardiac myocytes
Figure Lengend Snippet: Cystein 349 is an important region for the translocation of AT2R to the plasma membrane. a Amino acid sequence and the mutated cysteine residues in AT2R. The AT2R protein has four cysteine residues in intracellular region. The arrowhead indicates the position where GFP was inserted to make GFP-tagged AT2R (AT2R-GFP). b HEK293T cells were transfected with AT2R-WT, C70A, C71A, C319A, C349A mutants, respectively. Each mutant was treated with SNP (10 μM). Quantification of the mutants were identified by GFP protein. Na + -K + ATPase (NKA) was detected as a loading control for surface fraction. Bar graph represents the ratio of surface to total GFP protein. c Overexpressed AT2R-GFP (green) was localized in the cytoplasm in the HEK293T cell. The surface expression of AT2R-GFP was identified by WGA ( red ). The right panel showed the intensities of AT2R-GFP ( green ) and WGA ( red ) on yellowed dashed lines of the images. Scale bar 5 μm
Article Snippet: Fixed cells (not permeabilized) were incubated in blocking solution (5 % FBS in PBS) for 1 h, followed by an incubation with extracellular membrane targeting anti-rabbit AT2R polyclonal antibody (1:100, Alomone labs) for overnight at 4 °C.
Techniques: Translocation Assay, Sequencing, Transfection, Mutagenesis, Expressing, Whole Genome Amplification