COMMD8 promotes β-arrestin–mediated signaling of CXCR4. (A) IB analysis for Gαi activation induced by CXCL12 in control and Commd8Δ B cells. (B) CXCR4-mediated inhibition of cAMP production was assessed by the GloSensor reporter in vector-transfected control and COMMD8-deficient (sgCOMMD8) HEK293 cells expressing Flag-tagged CXCR4. The amounts of cAMP are plotted as fold increases of luminescence over the levels in unstimulated cells. Data are shown as the mean ± SD of triplicates and representative of three independent experiments. (C) Intracellular calcium responses to CXCL12 in control and Commd8Δ B cells. Responses are plotted as the ratio of Cal-520 to Fura Red fluorescence. Data are representative of two independent experiments. (D) IP assay for the recruitment of endogenous β-arrestin-2 to Flag-tagged CXCR4 in vector-transfected control and COMMD8-deficient (sgCommd8) 2PK-3 cells after stimulation with CXCL12. (E–H) IB analysis for the phosphorylation of ERK (E and G) and p38 (F and H) in Commd8Δ (E and F) and Arrb2−/− (G and H) B cells after stimulation with CXCL12. B cells from littermate Commd8f/+Mb1Cre/+ and Commd8f/fMb1+/+ (E and F) or Arrb2+/+ (G and H) mice served as the control. Error bars represent the mean ± SD of three (A and E–H) or four (D) independent experiments, and representative blots are shown. *, P < 0.05; **, P < 0.01; ns, not significant. The P values were obtained by two-tailed unpaired (A and D–H) or paired (B) t test. Asterisk indicates nonspecific bands (A, D, and H). coIP, coimmunoprecipitation.