Rac1 inhibition alters CXCR4 conformation. (A) HL60 cells were incubated with CXCL12 or the Rac1 inhibitory peptide at 4°C or 37°C and the CXCR4 surface signal (detected by mAb 44717) was measured by flow cytometry (n = 3). (B) Antibody-feeding experiments where HL60 cells were first stained with anti-CXCR4 mAb 12G5 and subsequently incubated with the Rac1 inhibitory peptide for different time periods. mAb signal was then measured by flow cytometry (n = 3). (C) Antibody-feeding experiments for which HL60 cells were first stained with anti-CXCR4 mAb 44717 and subsequently incubated with the Rac1 inhibitory peptide for 30 minutes or NSC23766 for 1 hour. mAb signal was subsequently measured by flow cytometry (n = 3). (D) Antibody-feeding experiments for which HL60 cells were first stained with the conformation-independent anti-CXCR4 mAb 4G10 and subsequently incubated with the Rac1 inhibitory peptide for 30 minutes or NSC23766 for 1 hour. mAb signal was subsequently measured by flow cytometry (n = 3). (E) HL60 cells were treated with NSC23766 and CXCR4 surface signal (detected by mAb 44717 or 4G10) was measured (n = 3). Bars show the mean fluorescence intensity determined by flow cytometry and expressed as percentage ± SEM compared with untreated or control conditions (*P < .05, **P < .01).