Figure 5

Staurosporine blocks PMA, but not PMA plus ionomycin, induced downregulation of CCR2 promoter activity. THP-1 cells were transfected with either 5 μg of vector alone (pGL3-basic; lane 1) or with 5 μg of the pGL3-1335 construct (lanes 2–7). In addition, each sample was also co-transfected with 2 μg of pRL-SV40 (renilla) to act as an internal control. Cells were then either left untreated (lanes 1–4) or pretreated with staurosporine (100 nM) for 2 hours (lanes 5–7). Next, the THP-1 cells were stimulated with a combination of PMA alone (lanes 3 and 6) or PMA plus ionomycin (lanes 4 and 7) for a further 46 hours. Subsequently, cell extracts were prepared and assayed for both luciferase and renilla activity. After normalization to the renilla control, the CCR2 transcriptional activity was determined relative to the pGL3-basic vector, which was arbitrarily assigned a value of 1. Similar results were obtained in two other experiments