Figure 2.

Determination of the Binding Affinity of CXCL12-AF647 at NLuc/CXCR4

(A–D) NanoBRET saturation ligand binding curves obtained in (A) membrane preparations from HEK293 cells exogenously expressing NLuc/CXCR4 (B) live HEK293 cells expressing genome-edited NLuc/CXCR4 (C) live HeLa cells expressing genome-edited NLuc/CXCR4 or (D) live HEK293 cells expressing genome-edited HiBiT/CXCR4 complemented with LgBiT. Cells or membranes were incubated with increasing concentrations of CXCL12-AF647 in the absence (black circles) or presence (white circles) of AMD3100 (10 μM) for 1 h at 37°C. Data shown are mean ± SEM and are representative of three or four independent experiments performed in duplicate for (A and B) and (C and D), respectively.

(E) Quantification of NLuc/CXCR4 expression by linear regression (F), as described in the STAR Methods, using membrane preparations made from HEK293 cells exogenously expressing NLuc/CXCR4 (NLuc/CXCR4 TG, black bar), HEK293 cells expressing genome-edited NLuc/CXCR4 (NLuc/CXCR4 HEK, gray bar), or HeLa cells expressing genome-edited NLuc/CXCR4 (NLuc/CXCR4 HeLa, white bar).

Data shown are (F) mean ± SEM or (E) representative of five individual experiments performed in triplicate (see Figure S2).