Journal: PLoS ONE

Article Title: Agonist-Directed Desensitization of the β 2 -Adrenergic Receptor

doi: 10.1371/journal.pone.0019282

Figure Lengend Snippet: (a)–(d) Confocal images of cells after immunostained with anti-β 2 AR. The cells were first stimulated with 2 nM epinephrine for 1 min (a), 2 min (b), 5 min (c) and 30 min (d). After epinephrine removal, the cells were further incubated in medium at 37°C for 29 min (a), 28 min (b), 25 min (c) and 0 min (d), respectively, such that equal time post stimulation was achieved for all. The staining was performed using anti-β 2 AR antibody and Alexa Fluor 488 goat anti-rabbit antibody. The nuclei were stained with DAPI. Confocal microscopy images were acquired on Zeiss confocal laser scanning microscope (oil-immersion, 63× objective). The green color is from the β 2 AR, the blue from the nuclei. (e) The agonist exposure time and Ro31-8220 sensitivity of pERK stimulated by epinephrine in A431 cells. The cells were incubated with the vehicle (0 min) or 2 nM epinephrine for a specific time (1 min, 2 min, 30 min), followed by further incubation at 37°C for another time to ensure all reach 30 min post simulation. Ro31-8220 when used was presented throughout the incubation. Equal amounts of cell lysate were separated by SDS-PAGE and analyzed for pERK by Western blotting. Equivalent gel loading was confirmed by probing with antibodies against GAPDH.

Article Snippet: Rabbit polyclonal anti-β 2 AR (#sc-569), mouse monoclonal anti-pERK1/2 (#sc-81492) and rabbit polyclonal anti-GAPDH (#sc-25778) antibodies were purchased from Santa Cruz Biotechnology Inc. (Santa Cruz, CA, USA).

Techniques: Incubation, Staining, Confocal Microscopy, Laser-Scanning Microscopy, SDS Page, Western Blot