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. 2016 Feb 3;291(19):9991–10005. doi: 10.1074/jbc.M115.712661

FIGURE 2.

FIGURE 2.

SDF1α and AM1241 did not compete for binding to CXCR4. MDA-MB-231 (A and B) or 293T cells (A) were plated in 96-well puncher plates and grown to a density of 75% prior to the experiment. To assign absolute affinity of each ligand for CXCR4 receptor, a competitive displacement assay was used as discussed by Misra et al. (62) using cold ligand AM1241 as a test compound along with [99mTc-MAS3]SDF1α and [99mTc-MAS3]AM1241 as radiotracers on the surface of MDA-MB-231 (CXCR4-positive) and 293T (CXCR4-negative) cell lines. Cells were washed twice with ice-cold 1× PBS, pH 7.4, and incubated for 20 min at 4 °C with 0.5 μCi of radiotracer in the presence or absence of the test compound. Cells were then washed thrice with 1× PBS, and the well contents were transferred directly to plastic tubes in γ counter racks. Well contents were counted on a Wallac Wizard 1470 γ counter. Experiments were performed at least thrice. Error bars represent S.E.