A pFastBac1 vector containing residues 2 to 332 of human ACKR3 with an N-terminal GP64 promoter, HA signal sequence, and C-terminal FLAG and 10× His tags was used for baculovirus expression (27). For expression in HEK293T cells, human ACKR3 was cloned into a pcDNA vector containing the Rluc3 gene (a gift from N. Heveker, Université de Montréal, Montréal, Québec, Canada) at the C terminus of the receptor. Constructs where 7 to 29 residues were removed from the receptor N terminus were obtained using the site-directed, ligase-independent mutagenesis (SLIM) protocol (45). For chemokine expression in Sf9 cells, CXCL11 and CXCL12 with their native signal sequences and C-terminal HA tags were cloned into a pFastBac1 vector containing a polyhedrin promoter. CXCL12P2G and CXCL12LRHQ mutant constructs were produced by site-directed mutagenesis using standard quick-change methods.

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