Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: β-Arrestin2 is a critical component of the GPCR–eNOS signalosome

doi: 10.1073/pnas.1922608117

Figure Lengend Snippet: Superresolution imaging of β-Arr2/eNOS in normal and injured SECs. SECs were isolated form normal (A) and injured (BDL, B) rat livers. β-Arr2 and eNOS were immmunolabeled with mouse monoclonal anti–β-Arr2 and rabbit polyclonal anti-eNOS antibody. β-Arr2 (Alexa Fluor 647, green) and eNOS (Alexa Fluor 568, magenta) were visualized in horizontal cross-section (X-Y), and in cross-section (X-Z) with an SR-200 inverted microscope (Vutara, Inc.) (60×/1.2 NA Olympus water immersion objective). Samples were imaged based on the SML biplane FPALM technology as described. Representative white field (Left) with the portion of the cell that is imaged at high resolution; X-Y section with eNOS labeled in magenta and β-Arr2 in green; and X-Z section images with β-Arr2 and eNOS colocalization appearing white from normal SECs (Upper) and injured SECs (Lower) are shown. (Scale bars, 1 μm.)

Article Snippet: Samples were imaged based on the single molecule localization (SML) biplane fluorescence photoactivation localization microscopy (FPALM) technology as described ( 27 , 28 ) and data were analyzed using Vutara SRX software (version 3.16).

Techniques: Imaging, Isolation, Inverted Microscopy, Labeling

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: β-Arrestin2 is a critical component of the GPCR–eNOS signalosome

doi: 10.1073/pnas.1922608117

Figure Lengend Snippet: ET-1 activation of β-Arr2 and eNOS. (A) SECs from normal (Left two panels) and BDL injured (Right two panels) rat livers were cultured for 24 h and exposed to ET-1 (20 nM) for 30 min. Cells were fixed and labeled with antibody to β-Arr2 (Upper red) and nuclei were labeled with DAPI (Lower, blue). Representative images (of more than 10 others) of single and merged channels are shown. (Scale bars, 10 μm.) (B) SECs isolated from normal rat livers (Left two panels) and BDL injured (Right two panels) rat livers were cultured for 24 h and exposed to ET-1 as in A. β-Arr2 and eNOS were immmunolabeled with anti–β-Arr2 and anti-eNOS antibodies. β-Arr2 (Alexa Fluor 647, green) and eNOS (Alexa Fluor 568, magenta) were visualized in horizontal cross-section (X-Y), in cross-section (X-Z) with an SR-200 inverted microscope (Vutara, Inc.) (60×/1.2 NA Olympus water immersion objective). Samples were imaged based on the SML biplane FPALM technology as described. Representative X-Y section (Upper) and X-Z (Bottom) section images from control (Left) and exposed to ET-1 (Right) are shown. (Scale bar, 1 μm.) (C) β-Arr2 and eNOS colocalization in normal and injured SECs as in B was quantified as in Materials and Methods; the changes in normal and injured SECs are presented graphically (1 for complete and 0 for no colocalization [n = 3/group]). (D) SECs isolated from β-Arr2 WT and KO mice were exposed to ET-1 (20 nM) for 30 min. Cell lysates were subjected to immunoblotting with the indicated antibodies. Specific bands corresponding to P-eNOS were quantified and are presented as the ratio of P-eNOS to eNOS, shown on the Right (n = 3/group). (E) eNOS enzymatic activity was measured in cell lysates treated as in D and NOS activity was normalized to that of control cells from β-Arr2 WT mice without ET-1 exposure and is presented graphically (the activity in SECs from β-Arr2 WT mice without ET-1 exposure was set at 100; n = 3/group). Statistical significance for C–E was evaluated by ordinary one-way ANOVA. Data are mean ± SD, *P < 0.05, **P < 0.01, ***P < 0.005 for differences between indicated groups; n.s., no significant difference.

Article Snippet: Samples were imaged based on the single molecule localization (SML) biplane fluorescence photoactivation localization microscopy (FPALM) technology as described ( 27 , 28 ) and data were analyzed using Vutara SRX software (version 3.16).

Techniques: Activation Assay, Cell Culture, Labeling, Isolation, Inverted Microscopy, Control, Western Blot, Activity Assay