Wet gels were autoradiographed at 70 C overnight. Electrophoretic mobility shift assay EMSA was performed according to the LightShift Chemi luminescent EMSA kit users instructions with minor modifications. especially Briefly, 5 ug of nuclear extracts were incu bated with 3 biotin labeled, purified double Inhibitors,Modulators,Libraries stranded oligonucleotide probes. The probes containing NFB consensus sites, NFBF were synthesized and labeled with biotin at the 3 end. After incubation for 30 min at room temperature, samples were separated on a pre run 5% polyacrylamide gel at 100 V for 90 min and then trans ferred Inhibitors,Modulators,Libraries to Zeta Probe GT nylon membrane. The probes were detected by HRP conjugated streptavidin and the bands were visualized by ECL reagents provided with the kit. The resultant bands were quantified using the imaging software Quantity One.

Comet assay The cells were rinsed twice with ice cold PBS and har vested, and the cell suspension was exposed to melpha lan on ice for 15 min. Immediately after treatment or after a 30 min recovery incubation at 37 C post treat ment, the cell suspension was stored on ice and an alka line comet assay performed using a Comet assay kit according to the Inhibitors,Modulators,Libraries manufacturers in structions with modifications. Co immunoprecipitation assay Cells were harvested by scraping and washed once with ice cold phosphate buffered saline. Cell pellets were resuspended and incubated in IP lysis buffer supple mented with protease inhibitor cocktail at a cell density of 107 cells ml on ice for 30 min. After centrifu gation at 12,000 g for 10 min at 4 C, the supernatant was collected as total cell lysate.

Protein concentration was determined by using the Bradford assay. Samples were pre cleared by incubating with protein A G agarose resin for 30 min on ice then coimmunoprecipitated for 3 h using APE1 antibody following the manufacturers Inhibitors,Modulators,Libraries instructions. After incubation, protein A G agarose resin was then added Inhibitors,Modulators,Libraries and incubated for 1 hour at 4 C. After washing 3 times with PBS containing protease inhibitor, the pellet con taining agarose beads together with binding proteins were mixed with sample buffer and incubated at 100 C for 5 min. The samples were then stored at 80 C or subjected to Western blotting analysis immediately.

Results The differential expression of APE1 in RPMI 8226 selleck products and U266 parental cell and their drug resistant cell lines RPMI 8226 LR5 and U266 LR6 To investigate the role of APE1 in melphalan resistance of multiple myeloma cells, we utilized melphalan resistant MM cell lines RPMI 8226 LR5 and U266 LR6 and their parental cells. The drug resistant statuses of all cell lines were validated by CCK 8 assay. The results suggested that the proliferation of 8226 LR5 and U266 LR6 cells are slightly inhibited by different doses of melphalan while the growth of the parental cell lines are significantly suppressed by the drug.