Production of CXCR4 mRNA by L540 cells.
2 × 106 cells/mL were incubated alone and with PMA–ion (15 ng/mL PMA + 500 ng/mL ionomycin), plastic-bound anti-CD30 agonistic mAb M67 (10 μg/mL), or BerH2 or anti-CD34 isotype-matched mAb as a control. At the time-points indicated, total mRNA was extracted and analyzed for CXCR4 and actin mRNA expression. Aliquots of cells were also pretreated with 20 μg/mL CHX before stimulation with PMA–ion and M67 mAb for 4 hours. Resting cells produced small amounts of CXCR4 (1.7 kb) and CXCR4-Lo mRNA (4 kb). At 4 hours, both PMA–ion and M67 strongly up-regulated CXCR4 mRNA. At 24 hours, PMA–ion–dependent CXCR4 mRNA transcription had reverted to basal levels, whereas the M67-dependent up-regulation persisted. CXCR4-Lo mRNA was not regulated, except in the case of pretreatment with CHX, which superinduced both CXCR4 and CXCR4-Lo mRNA in M67 and PMA–ion-stimulated cells. The experiment depicted is representative of 5.
