DMSO effect is independent of NF-κB binding and translocation. (A) Two percent (vol/vol) DMSO pretreatment has no effect on IκB proteolysis. Cells were stimulated with the indicated concentrations of TNF, in the absence or presence of 1 hour pretreatment with 2% (vol/vol) DMSO. Top panel, Western immunoblot with anti-IκB antibody; bottom panel, Western immunoblot with an anti-β–actin antibody (internal control). (B) Two percent (vol/vol) DMSO pretreatment has no effect on Rel A nuclear translocation. Cells were stimulated with 20 ng/mL TNF, in the absence or presence of 1 hour pretreatment with 2% (vol/vol) DMSO. Nuclei were purified over sucrose cushion and tested by Western immunoblot with anti-Rel A antibody. Sixty-five kD Rel A is strongly induced by TNF in the absence or presence of DMSO. A nonspecific band serves here as an internal control for protein loading (control). (C) Effect of TNF on NF-κB binding in the presence of DMSO. Cells were stimulated with increasing concentrations of TNF (indicated at top), in the absence or presence of 1 hour pretreatment with 2% (vol/vol) DMSO. Shown is EMSA analysis of nuclear extracts for binding to radiolabeled NF-κB. C1/C2 binding increases proportionally with TNF dose. 0.032 ng/mL TNF yields a 7-fold weaker signal than 20 ng/mL (360,351 v 2,645,248 arbitrary units [a.u.]). In contrast, 2% (vol/vol) DMSO pretreatment does not reduce NF-κB binding (2,691,962 a.u.). For nuclear extracts, stimulated with 20 ng/mL TNF, various concentrations of protein were used to determine assay linearity with protein input (compare lanes 7, 6, and 4). (D) DMSO inhibits IL-8 promoter induction independently of TNF-induced changes in NF-κB Rel A DNA binding. Luciferase induction of the IL-8 promoter by the indicated amounts of TNF. Triplicate cell cultures were transfected with the –162 IL-8/LUC reporter plasmid and an SV40/alkaline phosphatase plasmid as an internal control. Cells were unstimulated/stimulated with the indicated doses (in ng/mL) TNF. The fold-induction of Luciferase activity of stimulated cells is shown (P < .005 for all pairwise comparisons). 0.032 ng/mL TNF activates the NF-κB–dependent promoter 7-fold weaker than 20 ng/mL. Two percent (vol/vol) DMSO pretreatment reduces TNF-inducible promoter activity by 8-fold. (E) Abundance of TNF-inducible NF-κB Rel A binding is not changed by pretreatment with promoter-inhibitory doses of DMSO. Cells unstimulated/stimulated with 20 ng/mL TNF for 15 minutes in the absence or presence of 2% (vol/vol) DMSO. After treatment, nuclear extracts were analyzed for NF-κB binding by microaffinity isolation. Shown is the Western immunoblot with rabbit antihuman Rel A polyclonal antibody (NS, nonspecific).