Fig. 6. Nef-induced CCL2/MCP-1 expression in astrocytes is dependent on calmodulin. U251MG-parental (P) and U251MG-Nef_Bru cells (Nef) were treated with 50 µM W7 (+) or solvent (–) for 16 hours. Total RNA was isolated and analyzed with Multiprobe^TM ribonuclease protection assay (A) and RT-PCR (B). The ratios of the CCL2/MCP-1 to GAPDH intensities were determined and the values obtained from the solvent-treated U251MG-Nef cells were set to 100%. One representative experiment of three independent experiments is shown. (C) Astrocytic U251MG-Nef_Bru, -pNeo and -parental cells were incubated for 8 hours with W7 at concentrations indicated, and CCL2/MCP-1 protein in the supernatants was determined by ELISA. One representative experiment is shown. Data represent mean ± s.e.m. of duplicates.