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. 2021 May 12;4:569. doi: 10.1038/s42003-021-02070-9

Fig. 2. Internalization of CXCR4 is differentially affected by CXCL12, plerixafor, and AMD11070.

Fig. 2

a Antibody feeding experiment with FLAG-tagged CXCR4 and US28 in HEK293 cells. The cells were either immediately fixed (t = 0) or incubated at 37 °C for 30 min to induce internalization (t = 30) and then fixed. CXCL12 was added at 1 μM. Cell surface receptors are labeled with Alexa Fluor 488-conjugated secondary antibody (green, left column), while internalized receptors were labeled with Alexa Flour 568-conjugated secondary antibody (red, second column from left). Nomarski images are included to illustrate the cell outline (right column). Scale bar, 5 μm. Images show representative cells from three independent experiments. b Acute time-course internalization ELISA. HEK293 cells were transiently transfected with FLAG-tagged CXCR4 (white circle) or US28 (black square). For each receptor, the value at a given time point is corrected for background, normalized to the value at t = 0, and presented in percent with mean ± SEM shown. The experiment was performed at least four times in quadruples. c SNAP-tag CXCR4 constitutive internalization. Internalization was determined after preincubation with Tag-lite SNAP-Lumi4-Tb (donor) for 1 h at 4 °C (white circle) or 37 °C (black circle) and shown with mean ± SEM from at least four independent experiments performed in triplicates. d SNAP-tag CXCR4 agonist CXCL12 induced internalization in HEK293A cells. Cells were stimulated with increasing concentration of CXCL12; 0 nM (white circle), 0.1 nM (orange circle), 1 nM (yellow circle), 0.01 µM (blue circle), 0.1 µM (green circle) or 1 µM (black circle). Internalization was determined upon CXCL12 addition after 1-hour preincubation with Tag-lite SNAP-lumi4-Tb at 37 °C. Data are shown with mean ± SEM of triplicates from at least three independent experiments. e SNAP-tagged CXCR4 receptor internalization after stimulation with CXCL12 (black circle), plerixafor (red square), and AMD11070 (white square). The data were normalized to maximal internalization levels by CXCL12 for CXCR4 and presented with mean ± SEM of triplicates from at least six independent experiments.