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. 2013 Oct 6;288(47):34055–34072. doi: 10.1074/jbc.M113.512426

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© 2013 by The American Society for Biochemistry and Molecular Biology, Inc.

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FIGURE 1. — Characteristics of NELF-B modulation of GR induction properties of exogenous reporter. A and B, effect of exogenous NELF-B on A_max, -fold induction, PAA, and EC₅₀ of transfected reporter in U2OS cells. Triplicate wells of cells were transiently transfected with GREtkLUC reporter (100 ng in A, 12 ng in B) and the indicated amounts of chimeric NELF-B plasmid (A) or full-length NELF-B plasmid (B), without or with GR plasmid (0.5 ng in A, 0.2 ng in B), induced by steroid, and analyzed as described under “Experimental Procedures.” *, p < 0.05, **, p < 0.01 versus no NELF-B. y axis numbers are the quantitative values for the categories of the x axis (average ± S.E., n = 3 and 4 independent experiments in A and B, respectively). DM, Dex-Mes. C–E, modulation of GR activity with exogenous reporter upon reducing endogenous NELF-B. C, levels of NELF-B and control (LAD1) mRNAs in T47D cells stably transfected with shEGFP or shNELF-B RNA. Duplicate mRNA samples were prepared as described under “Experimental Procedures” and separated on agarose gels. The quantitative abundance was determined by qRT-PCR of the same original samples. D, representative dose-response curves for Dex induction of GREtkLUC in T47D cells stably transfected with shEGFP or shNELF-B RNA after transient transfection without or with GR plasmid. E, -fold changes in GR induction parameters from 5 independent experiments (± S.E.) in T47D cells stably transfected with shEGFP or shNELF-B RNA after transient transfection without or with GR plasmid. Thick horizontal dashed line represents no difference between shEGFP and shNELF-B cells. *, p < 0.02 versus no GR. F, graphic representation of domains of full-length rat GR and GR C terminus fused to GAL4 DBD. Cross hatching = GR DBD; shading = GAL4 DBD; striped = GR LBD. G, intracellular levels of NELF-B affect the EC₅₀ of gene induction by GAL/GR525C. Representative dose-response curves for Dex induction of GREtkLUC in T47D cells stably transfected with shEGFP or shNELF-B RNA after transient transfection without or with GAL/GR525C plasmid are shown. H, -fold changes in GR induction parameters from 7 independent experiments (± S.E.) in T47D cells stably transfected with shEGFP or shNELF-B RNA after transient transfection without or with GAL/GR525C plasmid. Thick horizontal dashed line represents no difference between shEGFP and shNELF-B cells. *, p < 0.05, ***, p < 0.0005 versus -fold difference = 1. I, binding of NELF-B to overexpressed GAL/GR525C. Cytosolic extracts of Cos-7 cells that had been transiently transfected with GAL/GR525C plus FLAG/NELF-B were treated with sodium sulfate, with or without EtOH (E) or Dex (D). Receptors were then analyzed for co-immunoprecipitation (IP) with FLAG/NELF-B using anti-FLAG antibody as described under “Experimental Procedures.” W, Western blot. J, NELF-B binds only to activated endogenously expressed GRs. A cytosolic solution of U2OS.rGR cells with endogenous GR and transiently transfected FLAG-tagged NELF-B was split in half, treated with sodium molybdate (to block activation) or sodium sulfate, and then incubated with EtOH (E), Dex (D), or RU486 (R) before being immunoprecipitated (IP) with anti-FLAG antibody as described under “Experimental Procedures.” W, Western blot.