Northern blot analysis of FPGS mRNA levels. RNA was blotted onto Zeta-Probe nylon membrane and hybridized with a [³²P]-labeled FPGS probe as detailed under “Northern blot analysis.” (A) RNA from parental CCRF-CEM and Molt-4 cells (lanes 1 and 4, respectively) as well as antifolate-resistant CEM-7OH MTX, CEM MTX^R10, and Molt-4-7OH MTX cells (lanes 2, 3, and 5, respectively). A discrete band with an expected size of 2400 bp can be observed for both parental cell lines (lanes 1 and 4), whereas the antifolate-resistant cells CEM-7OH MTX and MTX^R10 exhibit a smear spanning several hundreds of base pairs (lanes 2 and 3, respectively). (C) RNA from parental CCRF-CEM cells and their pIRES/FPGSΔexon10 transfectant (lanes 1 and 2, respectively) as well as the antifolate-resistant cell line CEM MTX^R10, its pcDNA3/hFPGS transfectant and its pIRES/FPGSΔexon10 transfectant (lanes 3 and 4, respectively). pIRES/FPGSΔexon10 is transcribed to yield an approximately 3550-bp-long operon including the FPGS gene, the IRES translation initiation sequence, and the EGFP gene. (B,D) The intensity of the FPGS transcript was normalized to the methylene blue staining of the 18S ribosomal RNA band.