Functional and physical interactions of β₂ARs with chemokine receptors. (A and B) Activation of Rac1 was analyzed by Western blotting in B and T cells isolated from spleens of WT (A) or Adrb2^−/− (B) mice at the indicated times after stimulation with CCL21 (2 µg/ml), CXCL12 (100 ng/ml), CXCL13 (1 µg/ml), or S1P (100 nM) in the presence or absence of clenbuterol (10 µM). (C) Rac1 activation was determined in B and T cells treated with clenbuterol in the absence of chemoattractants. Ratios of band density of GTP-bound Rac1 relative to that of total Rac1 were calculated and normalized to the ratio of the unstimulated (0 s) sample. The normalized ratios are shown under the lanes. Data are representative of more than two experiments for each cell type and condition. (D and E) Transwell migration of WT and Adrb2^−/− splenic B cells (CD19⁺; D) and CD4⁺ T cells (CD4⁺; E) in response to CCL21 (100 ng/ml), CXCL12 (10 ng/ml), CXCL13 (300 ng/ml), or S1P (100 nM), plus clenbuterol (at the indicated concentrations). Dashed lines indicate the levels of migration of WT (red) and Adrb2^−/− (blue) cells in the absence of chemoattractants and clenbuterol. Data are shown as mean ± range of duplicate wells and are representative of more than three experiments for each chemoattractant. (F) Representative confocal immunofluorescence images and analysis of 2PK-3 cells expressing Myc-tagged β₂ARs (green) and Flag-tagged chemoattractant receptors (red): CCR7 (top), CXCR4 (middle), and S1PR1 (bottom). Co-localization of the signals was quantified by measurement of overlapping pixel numbers as shown in the right (n = 95 for S1PR1; n = 99 for CCR7; n = 100 for CXCR4). DIC, differential interference contrast. Bar, 5 µm. Data are representative of three experiments. Bars indicate means. *, P < 0.05; ****, P < 0.0001 (one-way ANOVA with Bonferroni’s post-test). (G) Lysates of 2PK-3 cells expressing the indicated epitope-tagged receptors were subjected to immunoprecipitation (IP) with an anti-Flag antibody. Co-precipitated proteins were immunoblotted (IB) with an anti-Flag or anti-Myc antibody. Data are representative of three experiments.