Figure 6

GR site-specific phosphorylation, auto-induction, and potency in Dex-sensitive and –resistant CEM cells. A. GR ser211 phosphorylation and auto-induction. Naturally Dex-sensitive CEM-C7-14 (C7-14), Dex-resistant CEM-C1-15 (C1-15), and two resistant-to-sensitive revertant clones; CEM-C1-6 (spontaneous revertant, C1-6), and CEM-IV B9 (demethylated revertant, IV B9) were treated in triplicate for 16 hours with 100 nM Dex (D) or an equivalent volume of ethanol vehicle (C). Cell lysates were then analyzed for phospho-GR S211, total GR, and actin by immunoblot. Depicted is a representative blot from 3 independent samples done, all with nearly identical results. n = 3. The data shown are from a single gel, with irrelevant sections removed, for clarity. B. Dex/GR-dependent transcriptional activity. CEM; C1-15, C1-6, and IV B9 cells were transfected by electroporation with a GRE- dependent luciferase reporter plasmid. Cells were then divided into triplicate replicates and exposed to either ethanol vehicle (control) or 1 μM Dex for 24 hours after which time extracts were made and luciferase activity analyzed. A representative experiment is shown of average RLUs normalized to μg of protein in the cellular lysate. Error bars = 1 standard deviation from the mean. Total biological replicates, n = 2 for C1-15; 5 for C1-6; and 7 for IV B9.