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. 2014 Oct 15;25(20):3210–3221. doi: 10.1091/mbc.E14-03-0834

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© 2014 Cao, Yan, et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

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FIGURE 2: — AdcC transiently translocates from the cytosol to the cell membrane in an actin- and G protein–independent manner upon stimulation. (A) AdcC-YFP transiently associates with plasma membrane upon cAMP stimulation. adcC⁻ cells expressing AdcC-YFP were stimulated with 10 μM cAMP. (B) Kinetics of the time course. Means (n = 7) and SDs are shown. (C, D) cAMP-induced AdcC membrane translocation does not depend on the actin cytoskeleton. adcC⁻ cells expressing AdcC-YFP were treated with latrunculin B to depolarize the actin cytoskeleton and stimulated with cAMP. Means (n = 6) and SDs are shown. (E, F) cAMP-induced AdcC membrane translocation does not require Gβ. Translocation of AdcC-YFP in gβ⁻ cells was monitored by time-lapse confocal microscopy after cAMP stimulation. Means (n = 6) and SDs are shown. (G, H) cAMP-induced AdcC membrane translocation requires cAR1 signaling. Temporal changes in the levels of AdcC-YFP at the plasma membrane are shown as a time course for RI9 (car1-/car3-) cells expressing AdcC -YFP. Means (n = 6) and SDs are shown. (I, J) cAMP-induced PH_CRAC-GFP translocation appears normal in arrestin-null cells. adcB⁻C⁻ cells expressing PH_CRAC-GFP were treated with latrunculin B to depolarize the actin cytoskeleton and stimulated with cAMP. The levels of PH_CRAC-GFP at the plasma membrane are shown as a time course. Means (n = 6) and SDs are shown. Scale bar, 5 μm. In A and B, images were captured at 3.1-s intervals, and in B–J, images were captured at 2-s intervals; selected time points are shown.