Fig. 5

NS-398 effect on NF-κβ and IκB-α Expression in C4-2b Cells. C4-2b cells were cultured with NS-398 (10 µM) for 72 h. (A) Western blot analysis: total cellular protein isolated and separated by SDS-PAGE. Protein was transferred to PVDF and NF-κβ and IκB-α detected by Western blot analysis.IκB-α was detected using an antibody specific for the activated form. Blots are representative of data from two independent experiments. Lane 1, C4-2b DMSO control; lane 2, C4-2b, 10 µM NS-398. Upper bands represents NF-κβ p65, lower bands represents IκB-α. (B) Electrophoretic mobility assay (EMSA): double stranded oligonucleotides containing NF-κβ consensus sequence (GGGGACTTTCC) tandem repeats were labeled with ³²P. Nuclear extracts from DMSO control and NS-398 (10 µM) treated C4-2b cultures were mixed with labeled oligonucleotides and the resulting DNA-protein complexes separated by non-denaturing 6% polyacrylamide gel electrophoresis. NS-398 added to C4-2b cells increases NF-κβ binding to NF-κβ DNA consensus sequence oligonucleotide as determined by EMSA; lane 1, NS-398 (10 µM); lane 2, DMSO control; lane 3, NS-398 (10 µM) and MIF neutralizing antibody (1:200 dilution). Autoradiograph is representative data from two independent experiments. (C) Electrophoretic mobility assay (EMSA) NF-κβ specificity: reactions were performed as in Fig. 5 B except that addition of excess unlabeled competitor NF-κβ DNA consensus sequence oligonucleotide (800 ng) was added to NS-398 (10 µM) treated nuclear extracts. Lane (−) NS-398 (10 µM) treated nuclear extracts without addition of unlabeled competitor sequence; (+) NS-398 (10 µM) treated nuclear extracts with addition of unlabeled competitor sequence. Autoradiograph is representative of data from two independent experiments.