Figure 4

The PKC-inhibitor staurosporine blocks PMA, but not PMA plus ionomycin, induced downregulation of CCR2. (a). THP-1 cells were either untreated (lanes 1, 2, 7, 8, 13 and 14) or preincubated with 50 nM staurosporine (lanes 3, 5, 9, 11, 15 and 17) or 10 nM staurosporine (lanes 4, 6, 10, 12, 16 and 18) for 2 hours. Subsequently, the cells were stimulated with 50 nM PMA (lanes 2, 5, 6, 8, 11, 12, 14, 17 and 18) for a further 46 hours. Messenger RNA was then prepared and RT-PCR performed using primers for CCR1 (lanes 1–6), CCR2 (lanes 7–12) and GAPDH (lanes 13–18). M is a 100 bp DNA ladder. Similar results were obtained in three other experiments. (b). THP-1 cells were either untreated (lanes 1, 3, 5, 7, 9 and 11) or preincubated with 200 nM staurosporine (lanes 2 and 4, 6 and 8 and 10 and 12) for 2 hours. Subsequently the cells were stimulated with a combination of 1 nM PMA and 1 μM ionomycin (lanes 3 and 4, 7 and 8 and 11 and 12) for a further 46 hours. Messenger RNA was then prepared and RT-PCR performed using primers for CCR1 (lanes 1–4), CCR2 (lanes 5–8) and GAPDH (lanes 9–12). M is a 100 bp DNA ladder. Similar results were obtained in three other experiments.