Fig. 1
From: LPA1-mediated inhibition of CXCR4 attenuates CXCL12-induced signaling and cell migration
CXCR4 comes in close physical proximity to LPA1. A-G Representative confocal images to detect GPCR interactions in HEK293A cells using the BiFC assay. Cells were transfected with CXCR4-VN and CXCR4-VC (A), Flag-LPA1-VN and Flag-LPA1-VC (B), CXCR4-VN and Flag-LPA1-VC (C), Flag-LPA1-VN and CXCR4-VC (D), HA-μOR-VN and HA-μOR-VC (E), Flag-CXCR4-VN and HA-μOR-VC (F), or HA-μOR-VN and Flag-CXCR4-VC (G). Cell surface expression of CXCR4, LPA1, and μOR was visualized by staining cells with anti-CXCR4, anti-Flag, or anti-HA antibodies without permeabilization. Nuclei were stained with Hoechst 33342. H PLA was performed to visualize CXCR4 and LPA1 heteromerization in HEK293A cells. Cells transiently expressing Myc-CXCR4 and HA-LPA1, Myc-CXCR4 and HA-μOR, or Myc-CXCR4 alone were first stained with mouse anti-CXCR4 and rabbit anti-HA antibodies and then with anti-mouse and anti-rabbit secondary antibodies conjugated with plus and minus PLA probes. I The number of PLA dots per cell was counted using ImageJ software. Images containing a total of positive 30 to 90 cells were analyzed. Data represent the mean ± SEM of n = 3 independent experiments. Statistical significance was tested using unpaired two-tailed Student’s t test. ****P < 0.0001. J PLA was performed to visualize CXCR4 and LPA1 heteromerization in MDA-MB-231 cells. Representative PLA images for the detection of CXCR4, LPA1, and CXCR4-LPA1 heteromers using M1/M2 probe-conjugated anti-CXCR4 and mouse anti-LPA1 antibodies are shown. IgG isotype antibody was used for control experiments. K The number of PLA dots per cell was quantified using ImageJ software. For each independent experiment, 3 to 4 field images were used for quantification. Data represent the mean ± SEM of n = 3 independent experiments. Statistical significance was tested using unpaired two-tailed Student’s t test. ****P < 0.0001; ns, not significant. Scale bars: 20 μm