Figure 4

DAMGO alters surface expression of CXCR4 on TF-1 cells. (A) Total levels of CXCR4 remain unaltered in TF-1 cells upon DAMGO treatment. Western immunoblot analyses performed with TF-1 cell line whole cell lysates demonstrate the presence of glycosylated CXCR4 monomer at 45 KDa. In comparison to the untreated cells, DAMGO (1 and 10 μM)-treated cells exhibited a similar density of CXCR4, indicating that CXCR4 expression at the protein level does not change. The membrane was stripped and reblotted for human β-actin (43 KDa) as the endogenous loading control. (B) Treatment of TF-1 cells with DAMGO results in a shift in CXCR4⁺ cells to the low-expressing phenotype. Immunofluorescence flow cytometry analysis of nonpermeabilized TF-1 cells was performed to detect a change in surface expression of CXCR4 following DAMGO treatment. Immunofluorescence analysis for surface CXCR4 by flow cytometry demonstrated that TF-1 cells were distributed among three populations, non-expressing cells, low-expressing cells (lower mean fluorescence intensity [MFI]), and high-expressing cells (higher MFI). DAMGO pretreatment (1 and 10 μM) for 24 hours resulted in a shift in the relative proportion of CXCR4⁺ cells to the low-expressing phenotype, as is evident from the increase in the lower MFI peak and the reduction in the higher MFI peak. CXCR4 antibody isotype controls are shown at the bottom.