Figure 5. Detection of the interactions between CXCL12 and CXCR5_NT.

(A) The interaction between CXCL12 and the N-terminus of CXCR5 or CXCR3 was detected by a traditional Y2H, in which CXCL12 was fused with the activation domain of GAL4 (AD) as the bait, and CXCR5_NT or CXCR3_NT was fused with the binding domain (BD) of GAL4 as the preys. Only yeast expressing AD-CXCL12 and BD-CXCR5_NT could survive the selective medium, indicating the interaction between prey and bait. (B) CoIP of CXCL12 and CXCR5_NT. CXCL12 and CXCR5_NT-Fc or CXCR3_NT-Fc were co-transfected into the 293T cell line. Fc-tagged fusion proteins were precipitated by protein G beads, and the precipitates were separated by SDS-PAGE and blotted with rabbit anti-mouse CXCL12 IgG. The result shows that CXCL12 was precipitated by CXCR5_NT-Fc, but not by CXCR3_NT-Fc or Fc only, indicating an interaction between CXCR5_NT and CXCL12.