ChIP results obtained with the monoclonal antibody directed against hGRChIP assays were performed using HeLa cells, the monoclonal antibody directed against GR and optimized PCR primer sets for qPCR. The cells were treated either with ethanol (EtOH, used as a negative control) or triamcinolone acetonide (TA) for 4 hours prior to cell harvesting. ChIP was performed using sheared chromatin from 3 million cells and 5 μg of antibody. QPCR was performed with primers for the human metallothionein promoter (hMTIIA) and for exon 2 of the human myoglobin gene (hmyo ex2), used as a negative control. Figure 1 shows the recovery (the relative amount of immunoprecipitated DNA compared to input DNA) and the occupancy (ratio +/- control target). These results demonstrate the occupancy of the human metallothionein IIA promoter by GR.

Sandwich ELISA The specificity of the monoclonal antibody directed against hGR was assessed by sandwich ELISA. Figure 3A: schematic representation of the sandwich ELISA with the monoclonal antibody against hGR . Figure 3B: ELISA results using the monoclonal antibody against hGR at a concentration of 0.5 μg/ml. The figure shows an ELISA signal which is proportionally increasing with increasing amounts of recombinant hGR.

Western blot analysis using the monoclonal antibody against hGR Extracts from HeLa cells containing the indicated amounts of GR (from 30 to 240 fmol), and from 5x10e6 Raji or Molt cells were analysed by Western blot using the monoclonal antibody against hGR. Figure 4B. Western blot analysis of extracts from 300,000 HeLa cells with the monoclonal antibody against hGR (concentration 1 μg/ml).