Figure 6. Real-time monitoring of receptor ubiquitination using BRET2.
HEK293T cells were transfected with Ub-GFP2 (white bars) or (G75A,G76A)-Ub-GFP2 (filled bars) and (A) CXCR7-RLuc, CXCR7 ΔC-RLuc, or CXCR7 ST/A-Rluc, (B) CXCR4-RLuc, or (C) CXCR3-RLuc. BRET2 was measured 30 min after stimulation with 10−8 M of CXCL12 (CXCL11 for CXCR3) by addition of coelenterazine 400a and immediate read out. Results are expressed in Net BRET normalized to basal as described in Materials and Methods. Data represent the mean ± SEM of 3 experiments each performed in triplicate. **, p<0.01, and ***, p<0.001, by Student t test.