Journal: Scientific Reports

Article Title: Cysteine redox state regulates human β2-adrenergic receptor binding and function

doi: 10.1038/s41598-020-59983-4

Figure Lengend Snippet: Agonism of β2AR on human lung airway epithelial cells stimulates ROS generation. ( A ) CALU3 cells were preloaded with the intracellular ROS-sensitive fluorescent probe CM-H 2 DCFDA (10 µM) and stimulated with 10 μM ISO for 5 min or as otherwise shown, prior to fixation and confocal imaging. ISO stimulation elicited robust fluorescence, indicative of ROS generation, at the cell membrane, and this effect was blocked by pretreatment with alprenolol (10 µM). The overall degree of fluorescence was equivalent to that seen upon exogenous treatment with 1 mM H 2 O 2 . The upper panel is 40X image and the lower panel depicts further zoom on a single cell from that image. ( B ) Fluorescent microscopy of cells that were pretreated with the NADPH oxidase inhibitors apocynin (APO; 100 µM) and diphenyleneiodonium chloride (DPI; 10 µM) reveals marked decreases in the fluorescent intensity ( B ) and total number of fluorescent cells ( C ) in APO and DPI treated states. Images in ( B ) represent a 20X objective image capture with the top image representing FITC fluorescence, the middle image representing the respective transilluminated image, and the lower image representing the overlay. ( D , E ) Agonism of CALU3 cells with 1 μM ISO for 5 or 15 minutes oxidized cysteine residues on the β2AR enabling selective dimedone alkylation of these sulfenic acid residues with 5 mM dimedone (15 minutes) as can be seen by either β2AR immunoprecipitation followed by anti-Cys-dimedone immunoblotting ( D ) or Cys-dimedone immunoprecipitation followed by β2AR immunoblotting ( E ). Full length immunoblots are shown in Supplementary Fig. .

Article Snippet: FITC conjugated anti-mouse antibody was from Rockland Immunochemicals (Limerick, PA).

Techniques: Imaging, Fluorescence, Membrane, Microscopy, Immunoprecipitation, Western Blot