CD164 associates with CXCR4 and the integrins VLA-4 and VLA-5. (A) Coimmunoprecipitation of CXCR4 with CD164 and the integrins VLA-4 and VLA-5 in Jurkat cells in response to CXCL12 stimulation presented on fibronectin. Cells were stimulated with CXCL12 on fibronectin for 0, 1, 10, and 30 minutes. Immunoprecipitated CXCR4 was immunoblotted with mAbs to (i) CD164, (ii) VLA-4, (iii) VLA-5, (iv) ICAM-3, and (v) CXCR4. L = 50 μg total cell lysate. (B) Coimmunoprecipitation of CXCR4 and CD164 in CD133⁺ cells. Cells were stimulated with CXCL12 for 0, 1, and 10 minutes, and lysates were immunoprecipitated with anti-CXCR4 and immunoblotted with mAbs to (i) CD164, (ii) VLA-4, (iii) VLA-5, (iv) ICAM-3, and (v) CXCR4. (C) Single Z-stack confocal images of CD133⁺ cells in the absence (i, iii) and presence (ii, iv, v, vi) of CXCL12 for 10 minutes and coimmunolabeled with indicated antibodies. In the absence of CXCL12, CXCR4 and CD164 were equally distributed on the cell membrane. After CXCL12 stimulation, CD164, VLA-4, and VLA-5 redistribute to the leading edge of the cell with CXCR4, whereas ICAM-3 clusters to the rear and opposite pole. (D) Quantification of receptor redistribution in the absence (white bars) or presence (black bars) of CXCL12 stimulation. The number of cells that showed a polarized and colocalized distribution of CXCR4 with CD164, VLA-4, or VLA-5 was counted and expressed as a percentage of the total number of polarized cells observed, as defined by CXCR4 staining. Significant redistribution of CXCR4 with CD164, VLA-4, and VLA-5 was observed after CXCL12 presentation for 10 minutes (P < .05). ICAM-3 was counted as a control (P > .05).