MCP-1 mRNA expression and nuclear NF-κB. (A) RNA was isolated from five separate cultures of differentiated SH-SY5Y cells and used for quantitative RT-PCR. Expression of MCP-1 mRNA was normalized to the expression of GAPDH mRNA. MCP-1 mRNA expression was 3.0-, 3.2-, and 4.1-fold higher in vector-transfected, H63D HFE, and C282Y HFE cells, respectively, compared to cells expressing Wt HFE. (B) Nuclear fractions were isolated from four independent cultures of stably-transfected SH-SY5Y cell lines. Nuclear content of NF-κB (p50) was assessed by colorimetric assay and reported as optical density per milligram of nuclear protein. Results are expressed as mean ± SEM. No differences in NF-κB (p50) were found between any of the SH-SY5Y cell lines. n = 4. * and ** represent p < 0.05 and p < 0.01, respectively, compared to Wt HFE cells.