Effect of CD437 on NFkb and AP1 nuclear complexes as well as JNK kinase and protein levels in NB4 and NB4.437r cells.
(A) NB4 or NB4.437r cells (5 × 105/mL) were treated with vehicle, CD437 (10−6 mol/L), cycloheximide (CHX) (50 μM), PMA (1 μg/mL), or the indicated combinations of the compounds for 1 hour. Nuclear extracts were subjected to EMSA using radiolabeled oligonucleotide probes specific for NFkb, AP1, and SP1 transcription factors. Comp., cold oligonucleotide competitor. Supershift assay (right panel): nuclear extracts from cells treated with vehicle or CD437 (10−6 mol/L) for 1 hour were incubated with antibodies to the p65 component of the NFkb complex or with an irrelevant antibody (STAT1) of the same isotype before challenge with the radiolabeled oligonucleotide and subsequent EMSA. (B) NB4 or NB4.437r cells (5 × 105/mL) were treated with vehicle or CD437 (10−6 mol/L) for the indicated amounts of time. Cell extracts were prepared and JNK was immunoprecipitated with agarose-linked antibodies specific for the protein. Immunoprecipitates were incubated with γ32P-ATP in the absence (−) or the presence (+) of the JNK substrate gst-Jun. Equivalent amounts of the reaction mixtures were subjected to PAGE under denaturing conditions and subsequent autoradiography. To ensure that the same amount of JNK was present, Western blot analysis was performed on each immunoprecipitate using a second antibody specific to JNK-1. Data are representative of at least 2 independent experiments with similar results.
