G-CSF down-regulates CXCR4 mRNA expression in bone marrow myeloid cells. (A) Effects of cycloheximide (CHX) on CXCR4 protein levels in Gr1⁺ cells detected by immunoblotting with specific antibodies to CXCR4 and actin. Purified bone marrow Gr1⁺ cells were cultured (1 × 10⁶/mL) for 1 and 2 hours in medium alone (M) or with G-CSF (G, 100 ng/mL), with or without CHX (5 μg/mL). Total cell lysates were resolved by SDS-PAGE, transferred to nitrocellulose membrane, and immunoblotted (representative experiment of 3 performed). (B) Levels of CXCR4 mRNA in purified bone marrow Gr1⁺ cells immediately after purification and after culture with G-CSF (100 ng/mL, 30 minutes to 16 hours) measured by real-time RT-PCR analysis. The results are expressed as mean fold (± SE) mRNA change relative to mRNA levels detected before culture (results from 3 independent experiments each tested in triplicate).