Fig. 7. Myristoylation-deficient Nef_Bru does not increase CCL2/MCP-1 protein and mRNA production. U251MG-parental cells were transiently transfected with Nef_Bru, Nef_Bru-GG2AA or CAT. The cell culture supernatants were replaced with medium 8, 24 and 32 hours after transfection. (A) Alignment of the N-terminal amino acid sequence of Nef_Bru and Nef_Bru-GG2AA. (B). Simultaneous determination of Nef and GAPDH concentrations by western blotting. Cells were lysed after transfection as indicated. (C) CCL2/MCP-1 protein concentrations in the cell culture supernatants were determined using ELISA after transfection as indicated. Data represent mean ± s.e.m. of three separate transfection experiments. (D) Total RNA was isolated 48 hours after transfection and analyzed with RT-PCR. The ratios of the CCL2/MCP-1 to GAPDH intensities were determined and the values obtained from the Nef_Bru-transfected cells were set to 100%.