Effects of G-CSF on CXCR4 protein levels in murine bone marrow myeloid cells. (A) Typical representation of CXCR4 protein in cell lysates of freshly isolated Gr1⁺ cells purified from bone marrow cell populations detected by SDS-PAGE, transferred to nitrocellulose membranes, and immunoblotted with specific antibodies to CXCR4 (2B11 antibody). (B) Effect of the enzyme PNGase F (10 U/40 mg protein) on CXCR4 protein detected by immunoblotting (2B11 antibody). (C) Time-dependent reduction of Gr1⁺ cell–associated CXCR4 induced by G-CSF. Cells lysates were prepared from purified Gr1⁺ bone marrow cells either immediately after purification or after culture in medium alone (M) or with G-CSF (G, 100 ng/mL). Samples were immunoblotted with anti-CXCR4 antibodies (2B11), stripped, and reprobed with antibodies to beta-actin (representative experiment of 5 performed). (D) Appearance of CXCR4-related bands after 18-hour incubation of electronically sorted Gr1⁺ cells (> 95% purity) in medium only (M) or with G-CSF (G, 100 ng/mL). Bottom panel reflects actin-related bands upon reprobing with anti–beta actin antibodies. (E) Levels of CXCR4 protein in Gr1⁺ cells immediately after purification and after culture (1-18 hours) with medium only or with 100 ng/mL G-CSF measured by band intensities relative to actin. The results are expressed as percent CXCR4 measured in Gr1⁺ cells immediately after purification (mean ± SD of 3-6 separate experiments; 1-hour time point: mean of 6 experiments; all other time points: mean of 3 experiments). *P < .05; **P < .1 (medium vs G-CSF).