Fig. (4) Real time PCR determination of GARFT mRNA expression (A) and western blotting assay for GARFT protein expression levels (B) indicate that the acquisition of resistance to AG2034 is associated with increases in GARFT mRNA and protein levels. Cells (1 x10⁶ cells/flask) were seeded and allowed 24 h to adhere to plate in 0HPX media with (Resistant cells) or without (Parental cells) 50 nM AG2034. Thereafter, culture media was renewed appropriately with fresh media for a further 24 hours before total RNA or protein extraction. The figure represents: drug naïve parental DU-145 cells (Parent), parental cells treated with drug for 24 hours (Parent+D) and drug-resistant DU- 145 cells (Resistant). Data in panel A represent means ± S.D. of three independent determinations. The results in Panel B are representative of three individual blots.