Fig. 5
From: Functionally distinct and selectively phosphorylated GPCR subpopulations co-exist in a single cell
Subpopulations of β2AR are spatially segregated between dendrites and soma in hippocampal neurons. Primary hippocampal neurons expressing FLAG-β2AR at 18–21 days in vitro (DIV, mature, a) or 6–8 DIV (immature, b) were treated with 1 μM ISO for 5 min (a) or 10 min (b). Neurons were stained with phospho-specific antibodies against S261/262 (PKA-pβ2AR) and S355/356 (GRK-pβ2AR) as indicated to show phosphorylated β2ARs in soma, and proximal and distal dendrites (Scale bar, 2 μm. n = 25 and 20 cells, respectively, three independent experiments). Representative SIM images in a and b were from one single neuron. c Numbers of fluorescent objects per 20 μm2 regions were quantified with ImageJ in 18–21 DIV mature neurons (PKA-pβ2AR, soma 29.6 ± 1.7, proximal dendrites 42.6 ± 2.2, and distal dendrites 38.6 ± 1.8; GRK-pβ2AR, soma 50.4 ± 2.7, proximal dendrites 14.9 ± 0.7, and distal dendrites 5.1 ± 0.6). d Numbers of fluorescent objects per 20 μm2 regions were quantified with ImageJ in 6–8 DIV immature neurons (PKA-pβ2AR, soma 31.4 ± 1.7 and dendrites 42.6 ± 1.8; GRK-pβ2AR, soma 45.1 ± 2.0, and dendrites 36.8 ± 1.5). Error bars denote s.e.m., multiplicity adjusted P values are computed by one-way ANOVA followed by Tukey’s test between indicated groups
