Both cell lines were maintained in RPMI 1640 supplemented with 1 mM sodium pyruvate and 10% fetal DZNeP FDA bovine serum. Adenoviral vectors e pressing B galactosidase, eIF5A1, and eIF5A1K50A were constructed and propagated as described. For adenovirus mediated transfection, cells were seeded at 100,000 cells per well on a 24 well tissue culture plate and incubated with adenovirus constructs at multi plicities of infection, the ratio of the number of infectious viral particles to the number of target cells, ranging from 5 to 80 in medium containing 0. 5% FBS. Four hours later, the media was replaced with growth media or growth media containing 10 uM of the inhibi tors U1026, SB203580, SP600125, or 30 uM of pifithrin. Dimethylsulfo ide was included as a vehicle control.
SDS PAGE and western blotting Cell lysate was prepared in lysis buffer followed by brief sonication. Protein concentration was quantified using the Bicinchoninic Acid Kit. One to ten micrograms of protein was separated by SDS PAGE and western blot analysis was performed by incubating with primary antibodies for either one hour or overnight at 4 C. After incubation with HRP conjugated secondary anti bodies, the antibody protein comple es were visualized using enhanced chemiluminescence. Densi tometry analysis was performed using TotalLab TL100 vs2006 software. In order to distinguish between the different post translational modification states of eIF5A, two dimensional gel electrophoresis followed by west ern blot analysis using eIF5A antibody was performed as described.
Briefly, cell lysates were harvested in cold lysis buffer, loaded on Immobiline Drystrips followed by electrofocusing with Ethan IPGphor II using the following program 500 V 0. 5 hr, Grad 1000 V 0. 5 hr, Grad 5000 V 1. 5 h, 5000 V 6 hr, 500 V 5 hr. Proteins were then fractionated on a 12% SDS PAGE gel, transferred to a PVDF membrane, and eIF5A post translational modified forms were identified by blotting with an antibody against eIF5A1. RT qPCR Total RNA was isolated from cells infected with adeno viral constructs using the GenElute Mammalian Total RNA Miniprep Kit. Reverse transcrip tion was performed on 1. 2 micrograms of total RNA using AMV reverse transcriptase according to the manufacturers instructions. PCR reac tions contained 500 nM of each primer, 1�� of iQ SYBR Green Supermi , and 1 uL of cDNA.
Real time PCR was performed in a MiniOpticon Real Time PCR De tection System for 40 cycles using glyceralde hyde 3 phosphate dehydrogenase as a reference Apoptosis assays Apoptosis was quantified by labeling cells with Anne in V FITC and propidium iodide using the FITC Anne in V Apoptosis Detection Kit II, according to the manu Brefeldin_A facturers instructions, followed by analysis on a BD FACSVantage SE system with an argon laser source.