Figure 6

Inhibition of IL-1β-induced NF-κB activation in glEND.2 cells by antileukoproteinase. (a) The left panel shows an electrophoretic mobility-shift assay (EMSA) performed with nuclear extracts from unstimulated control cells (lane 4) and IL-1β-stimulated glEND.2 cells that had been preincubated in either the absence (lane 1) or the presence of antileukoproteinase (ALP; lane 2) or human serum albumin (hSA; lane 3) as an irrelevant control protein. Pretreatment of the glEND.2 cells with ALP was associated with a clearly decreased nuclear translocation of NF-κB reflected by the weakened intensity of the corresponding band shifts of the radiolabelled NF-κB-specific oligonucleotides. For comparison a concomitantly developed EMSA from the same experiment shows the electrophoretic mobility of the constitutively active transcription factor Oct-1 (right panel; lanes loaded as described for the left panel). (b) The densitometric evaluation of an independently performed EMSA experiment depicts the difference in NF-κB signal intensities between unstimulated control (- IL-1β control) and IL-1β-stimulated glEND.2 cells preincubated with either 5 μM ALP or 5 μM hSA. As a quantitative measure of the nuclear translocation of NFκ-B the figure shows the relative DNA-binding activity of NF-κB. This parameter represents the numerical proportion between NF-κB-binding and Oct-1-binding activity in c.p.m. The protein binding to the radiolabelled specific probe for the constitutively active transcription factor Oct-1 served as an internal standard for normalization of the nuclear extracts. (c) An additional specificity control for the NF-κB EMSA. Before electrophoretic separation, nuclear extracts from IL-1β-stimulated glEND.2 cells were incubated not only with the radiolabelled NF-κB but also with two different concentrations of unlabelled competitor probes. The results of the blocking experiments of the NF-κB EMSA with an unlabelled NF-κB-probe (Inh) (showing blocking at 10-fold (*) and 100-fold (^#) molar excess) and a mutant NF-κB probe (Mut) that did not suppress the NF-κB signal at 10-fold and 100-fold molar excess, respectively, are shown. Lane 1 was loaded with a sample without competing probe as in (a).