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Cyclosporin A Affects Signaling Events Differentially in Human Gingival Fibroblasts

Department of Pathology, Box 357470, University of Washington School of Medicine, Seattle, WA 98195-7470, USA;

View larger version (62K): [in a new window]   Figure 1. Gel shift assay to determine DNA-binding activity of NFAT and NFB transcription factors. Nuclear extracts were obtained from human gingival fibroblasts exposed to 10 ng/mL TNF- in the presence or absence of 10 µg/mL cyclosprin A. Five µg of nuclear proteins were incubated with [32P]ATP-labeled oligonucleotide probes, separated as described in the "METHODS", and visualized by autoradiography. For competition, unlabeled nucleotides were added at 100-fold molar excess. Arrows indicate shifted complexes. A, NFAT; B, NFB.

View larger version (35K): [in a new window]   Figure 2. Western blot analysis of immunoprecipitates obtained with the use of anti-p38 antibody. Human gingival fibroblasts were treated with 10 ng/mL TNF- and 10 µg/mL cyclosporin A. Cell lysates were subjected to immunoprecipitation, separated by SDS-polyacrylamide gel electrophoresis, and transferred to nitrocellulose membranes. Blots were probed with antibodies to phosphorylated (top panels) and unphosphorylated (bottom panels) enzymes. Incubation with TNF- was for 20 min (lanes "a" and "b") or for 4 hrs (lanes "c" and "d").

View larger version (42K): [in a new window]   Figure 3. Western blot analysis of immunoprecipitates obtained with the use of antibody to unphosphorylated JNK. Cells were treated with cyclosporin A and TNF-, and cell lysates were processed as described in Fig. 2. The blots were probed with antibodies to phosphorylated (top panels) and unphosphorylated (bottom panels) enzymes. Incubation with TNF- was for 20 min (lanes "a" and "b") or for 4 hrs (lanes "c" and "d").

View larger version (59K): [in a new window]   Figure 4. AP-1 activity of human gingival fibroblasts treated with and without 10 µg/mL cyclosporin A and 10 ng/mL TNF- or PDGF. (A) Gel shift assay; 5 µg of nuclear proteins were incubated with labeled [32P]ATP-labeled oligonucleotide probe, separated, and visualized by autoradiography, as described in the "METHODS". For competition, unlabeled nucleotide was added at 100-fold molar excess. Arrows indicate shifted complexes. (B) Luciferase reporter activity normalized for ß-actin Renilla luciferase control. Mean ± SD of triplicates is shown (p < 0.025).