Cooperative transcriptional activation of MBP gene expression in NIH 3T3 cells by C/EBPε₃₂, GATA-1, and PU.1.

(A) NIH 3T3 cells were seeded in 6-well plates at approximately 70% confluence and were transfected with the combinations of expression vectors indicated below the graph. The total amount of expression vector was kept equal in each transfection with the addition of empty vector. The combinations of expression vectors and the ratio of FOG expression vector to GATA-1 expression vector are indicated on the x-axis. At 24 hours after transfection, the cells were lysed in TRIzol reagent, total RNA was isolated and treated with DNaseI, and cDNA was synthesized. Quantitative RT-PCR using SYBR Green was performed in triplicate for each sample. The levels of MBP expression were normalized to 18S. Results are presented as fold changes (± SD) compared with cells transfected with empty vector. (B) Southern blot analysis of the amplification products from single (−, ε, G, and P) or 3 expression vector (ε+G+P)–transfected NIH 3T3 cells. PCR was performed for MBP (33 cycles) and GAPDH (25 cycles). − indicates empty; (ε), C/EBPε; (P), PU.1; (G), GATA-1; (F), FOG.