Fig. 1
From: Functionally distinct and selectively phosphorylated GPCR subpopulations co-exist in a single cell
PKA- and GRK-pβ2ARs are spatially segregated on the plasma membrane. a, b SIM imaging shows total β2ARs, and PKA- and GRK-phosphorylated β2ARs, which were stained with anti-FLAG, anti-S261/262 (PKA-pβ2AR), and anti-S355/356 (GRK-pβ2AR) specific antibodies, respectively, in HEK293 cells expressing FLAG-tagged β2AR before stimulation (a) or after 30 s of stimulation with 1 μM ISO (b). Scale bar, 2 μm. Representative of n = 5, 4, 19, 11, and 13 cells, respectively, three independent experiments. c The overlap between two different staining from images in b was evaluated by Pearson’s correlation coefficient (n = 19, 11, and 13 cells, respectively). d Quantification of two-color overlap was evaluated in HEK293 cells after 1 min of stimulation with 1 μM ISO using the same method in b (n = 13, 12, and 11 cells, respectively, three independent experiments). e FLAG-β2ARs expressed in HEK293 cells were stimulated with 1 μM ISO for 30 s, and cell lysates were subjected to immuno-isolation with anti-pS355/356 (GRK-pβ2AR) specific antibody (1st immunoprecipitate, IP). The remaining β2ARs in the supernatant were isolated with anti-FLAG antibody (2nd IP). Total β2AR were immuno-isolated directly with anti-FLAG antibody (total IP). The β2AR in total IP and sequential IPs were resolved with 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. Molecular weight markers (in kDa) are indicated on the left. Error bars denote s.e.m.; multiplicity adjusted P values are computed by one-way ANOVA followed by Tukey’s test between indicated groups
