Figure 3

Transcription factor binding profile of undifferentiated and neutrophil-like PLB-985 cells. (A) Undifferentiated or DMSO-differentiated PLB-985 cells (day 5) were incubated for 15 min at 37°C in the absence ("-") or presence of 100 ng/ml LPS or 100 U/ml TNFα, and nuclear extracts were prepared and analyzed in EMSA (1 μg/lane) using NF-κB, C/EBP, or AP-1 oligonucleotide probes. (B) Undifferentiated or DMSO-differentiated PLB-985 cells (day 5) were incubated for 15 min at 37°C in the absence ("-") or presence of 100 U/ml IFNγ, 1 nM GM-CSF, or 1000 U/ml G-CSF, and nuclear extracts were prepared and analyzed in EMSA (1 μg/lane) using GRR or hSIE oligonucleotide probes. (C) Nuclear extracts from TNF-stimulated granulocytic PLB-985 cells (day 5) were incubated for 30 min in binding buffer in the presence of antibodies to p50, p52, c-Rel, RelA, or an isotype-matched antibody ("IgG"), or in the presence of a 10- or 25-fold molar excess of unlabeled NF-κB oligonucleotide probe ("cold"), or in the presence of a 25-fold molar excess of unlabeled NF-κB probe featuring a mutated NF-κB site ("mut"), prior to the addition of labeled NF-κB probe and subsequent EMSA analysis. (D) Nuclear extracts from IFNγ-stimulated granulocytic PLB-985 cells (day 5) were incubated for 30 min in binding buffer in the absence ("-") or presence of antibodies to STAT1, STAT3, or an isotype-matched antibody ("IgG"), prior to the addition of labeled hSIE/m67 probe and subsequent EMSA analysis. Experiments shown in this figure are representative of at least three.