Fig. 2
From: Functionally distinct and selectively phosphorylated GPCR subpopulations co-exist in a single cell
PKA- and GRK-pβ2AR undergo distinct membrane trafficking. a FLAG-β2ARs expressed in HEK293 cells were stimulated with ISO for indicated times. Confocal imaging shows PKA- and GRK-phosphorylated β2ARs, which were stained with anti-pS261/262 (PKA-pβ2AR) and anti-pS355/356 (GRK-pβ2AR) antibodies, respectively. Scale bar, 5 μm. Representative of n = 16 and 15 cells, respectively, three independent experiments. b Numbers of fluorescent objects in each cell in a were quantified with ImageJ (n = 19, 16, and 15 cells, respectively, three independent experiments). Error bars denote s.e.m.; multiplicity adjusted P values are computed by one-way ANOVA followed by Tukey’s test between indicated groups. c Immuno-isolation of PKA- and GRK-phosphorylated β2ARs in HEK293 cells using same procedure as Fig. 1d after stimulation with 1 μM ISO for 10 min. The β2AR in total IP and sequential IPs were resolved in SDS-PAGE, and probed with anti-FLAG, anti-pS261/262 (PKA-pβ2AR) and anti-pS355/356 (GRK-pβ2AR) antibodies, respectively. Representative of three independent experiments. d FLAG-β2ARs expressed in HEK293 cells underwent cell surface biotin labeling and then were stimulated with ISO (1 nM or 1 μM) for 10 min. The biotin-labeled proteins on the plasma membrane were pulled with streptavidin beads, and the leftover biotin-labeled proteins in endosome were isolated by a second precipitation with streptavidin beads. Membrane (M) and endosome (E) fractions were resolved in Western blot with antibodies against FLAG, pS261/262 (PKA-pβ2AR), and pS355/356 (GRK-pβ2AR), showing separation of GRK- and PKA-phosphorylated subpopulations of β2AR. Representative of three independent experiments. Molecular weight markers (in kDa) are indicated on the left
