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Figure 2. The role of IGF-1 signaling on PDLF vs. GF. Human PDLF and GF were cultured in six-well plates and were treated with IGF-1 and LY294002 as described in MATERIALS & METHODS. (A-D) We used flow cytometry to detect the annexin V-FITC-generated signal at 488 nm (FL1-H). The shadowed area represents fluorescent intensity distribution of PDLF cells. The area under the bold line represents fluorescent intensity distribution of GF cells. (E) The extent of DNA fragmentation was assessed by the diphenylamine (DPA) assay as described in MATERIALS & METHODS. (F) Cell lysate was analyzed by Western blot. Phosphorylated proteins were detected by the use of antibodies against phospho-PKB (Ser473) and phospho-Bad (Ser112). Antibodies against PKB or Bad were also used for detection of the total amount of PKB or Bad in the lysate. (G) We used an ELISA system (R&D Systems, Minneapolis, MN, USA) to determine the concentration of active caspase 3 by measuring the optical density at 450 nm. All bar graphs represent Mean ± SD, n = 4. *p < 0.05, t test, difference between PDLF and GF. D, serum-free DMEM. F, DMEM containing 10% fetal bovine serum. I, 10-8 M IGF-1. LY, 10-6 M LY294002.
