Sub-optimal concentrations of PMA, together with a modest calcium signal, also modulate CCR2. (a). THP-1 cells were either unstimulated (lane1) or treated with PMA 1 nM (lane 2) or 50 nM (lane 3) for 48 hours. Alternatively, the cells were treated with increasing concentrations of the calcium ionophore ionomycin alone (lanes 4–6) or in combination with PMA 1 nM (lanes 7–9) also for 48 hours. Messenger RNA was then prepared and RT-PCR performed using primers for CCR1 (upper panel), CCR2 (middle panel) and GAPDH (lower panel). M represents markers, which are a 100 bp ladder. Similar results were obtained in four other experiments. (b). THP-1 cells were either left untreated or stimulated with PMA (1 nM) and ionomycin (1 μM) for the times indicated. Subsequently the cells were stained for expression of CCR1 (left panel) or CCR2 (right panel) and analyzed by flow cytometry.