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Fig. 2. Level of expression of human reduced folate carrier (hRFC) and human proton-coupled folate transporter (hPCFT) protein and mRNA as well as activity of the SLC19A1 and SLC46A1 promoters in preconfluent, confluent, and postconfluent Caco-2 cells. A and D: Western blot analysis was performed using membranous fraction (150 µg) isolated from preconfluent, confluent, and postconfluent Caco-2 cells and specific anti-hRFC and anti-hPCFT polyclonal antibodies. Using an enhanced chemiluminescence kit (Amersham, Arlington Heights, IL), immunoreactive bands were detected as described in MATERIALS AND METHODS. Note that the lane images show proteins detected on a representative single blot; however, they were rearranged for clarity of presentation. B and E: quantitative real-time PCR was performed using hRFC (B) and hPCFT (E) gene-specific primers and total RNA (5 µg) from preconfluent, confluent, and postconfluent Caco-2 cells. Real-time PCR was performed as described in MATERIALS AND METHODS. Data represent means ± SE of at least 3 independent sets of experiments and were normalized relative to β-actin and calculated using a relative relationship method supplied by the iCycler manufacturer (Bio-Rad, Hercules, CA). Note that the level of expression of hRFC and hPCFT in preconfluent cells was set at 1 for each figure and the expression during differentiation is in relation to that level; therefore the results are not representative of the levels of hRFC compared with hPCFT (hPCFT mRNA expression is  3-fold higher than hRFC at all 3 stages of differentiation). C and F: activity of full-length hRFC promoter B (C) and hPCFT promoters (F) in pGL3-Basic was determined following transient expression in preconfluent, confluent, and postconfluent Caco-2 cells. Luciferase activity was determined and normalized relative to the activity of simultaneously expressed Renilla luciferase as described in MATERIALS AND METHODS. The results are expressed relative to the PGL3-Basic vector set at 1. Data represent means ± SE of at least 3 independent sets of determinations.
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