Fig. 3. CCL2/MCP-1 expression is increased in nef_Bru-expressing U251MG cells. (A) Astrocytic U251MG-Nef_Bru (clones 4/4.1 and 4/4.2), -Nef_TH, -pNeo and -parental cells were seeded at a density of 2x10⁵ or 4x10⁵ cells/ml, incubated for 8, 16, 24 or 36 hours, and supernatants were subsequently analyzed with CCL2/MCP-1 ELISA. Fold inductions were calculated relative to the CCL2/MCP-1 protein concentrations determined in the supernatants of U251MG-parental cells. Data represent mean ± s.e.m. from at least six independent experiments; *, P<0.05. (B) Kinetic analysis of CCL2/MCP-1 protein secretion. Cells were incubated for periods as indicated and CCL2/MCP-1 protein concentrations determined by ELISA in the cellular supernatants. One representative experiment is shown. Data represent mean ± s.e.m. of duplicates. (C) Cells were incubated for 4 hours in the presence of monensin and subsequently stained intracellularly with PE-labeled mAb to CCL2/MCP-1 (black outline) or PE-labeled isotype-matched control antibody (gray). The ratio of the mean fluorescence intensities from CCL2/MCP-1-stained to isotype-stained cells is indicated in the upper right corner of each plot. (D) Total RNA was isolated from astrocytic U251MG-Nef_Bru (clones 2/8.2, 4/4.1 and 4/4.2), -Nef_TH and -pNeo cells, and analyzed by Multiprobe^TM RNase protection assay using biotinylated hCK-5 as probe.