FIGURE 2.

Protein expression levels of reduced folate carrier (RFC), thymidylate synthase (TS), and dihydrofolate reductase (DHFR) and their correlation with N‐Myc expression. A, Cell lysates were subjected to western blot analysis with anti‐N‐Myc, anti‐RFC, anti‐TS, anti‐DHFR, and anti‐β‐actin Abs. B, Protein expression levels of RFC, TS, and DHFR (normalized against β‐actin) in 6 MYCN‐amplified and 4 MYCN nonamplified (Nonamp) neuroblastoma cell lines. C, Flow cytometric analysis of fluorescein methotrexate (F‐MTX) incorporation in 2 MYCN‐amplified (IMR‐32 and KELLY) and 2 MYCN nonamplified (SH‐SY5Y and SK‐N‐FI) cells. Cells were incubated with 0, 1, 3, and 10 μmol/L F‐MTX for 2 h and washed with fresh culture medium without F‐MTX for 30 min. A total of 20 000 cells were analyzed by flow cytometer