text: Inhibition of transcription factors
  To inhibit translocation and binding of transcription factors to the IL-1RA promoter, PBMC were incubated for 1 h with each of the following substances (concentrations were based upon 50% inhibition): 50 mug/ml of CAPE (caffeic-3,4-dihydroxycinnamic-acid-phenyl-ester), (Biomol, Hamburg, Germany); 400 ng/ml cyclosporin A (Calbiochem-Merck Biosciences, Bad Soden, Germany); 10 mug/ml cycloheximide (Santa Cruz Biotechnology) prior to incubation with GP. Band shifts were determined after 1 h incubation with GP as described before, including mutated oligos for NFIL-6 (TTA CAA CAG TGG ATT GCG ACA CTT AGT GGG) and NFATP2/3 (GGC GCA GAA AAG GTA AAA TAT TTA CTA TCT) binding sites within the IL-1RA promoter. PBMC RNA was isolated with an RNeasy kit (Qiagen, Germany) and messenger RNA was transcribed into cDNA with Reactin Ready First Strand kit (Biomol) and analysed for IL-1RA and GAPDH transcripts by PCR with a HotStart "sweet" PCR mastermix (Biomol) (95degreesC, 30 sec, 55degreesC, 30 sec, 72degreesC, 30 sec, 25 cycles for IL-1RA and 94degreesC for 30 sec, 50degreesC for 30 sec and 72degreesC for 45 sec, 25 cycles for GAPDH) following 18 h incubation with GP. IL-1RA protein was measured in the supernatant by ELISA after 24 h incubation with GP. 
  