Figure 6
Figure 6. Enhancement of rRNA synthesis by co-expression of TIF-IA with Ebp1. (A) Effects of Ebp1 depletion on rRNA synthesis in T cells. Cultured T cells were transfected with SCR or siEbp1 (20 nM) for 36 hours. 5′ETS pre-rRNA levels and RNA labeling (left) and ChIP assay with Pol I antibody and western blot (right). (B) Effects of Ebp1 depletion on TIF-IA localization; 293T cells were transfected with SCR or siEbp1 (20 nM) for 36 hours. Cells were co-stained with anti–TIF-IA and anti-UBF antibodies (left). rDNA was labeled with a rDNA probe as described in “Methods.” Fluorescence intensity was measured along the line through 3D pictures on the left (right). (C) Effects of Ebp1 depletion on TIF-IA binding with Pol I in primary T cells. Cells were transfected with SCR or siEbp1 (20 nM) for 36 hours. Nucleoli were isolated (see “Methods”) and nucleolar or whole cell lysates were immunoblotted for TIF-IA, Ebp1, and NPM1 (left), whereas cell lysate was incubated with anti–TIF-IA and the precipitate immunoblotted with anti-Pol I antibody (right). (D) Effects of co-overexpression of TIF-IA and Ebp1 on rRNA synthesis. Jurkat T cells were co-transfected with GFP-Ebp1 and vector control or Myc–TIF-IA for 24 hours. 5′ETS pre-rRNA and RNA labeling (left) and Pol I binding by ChIP assay and western blot (right). (E) Effects of TIF-IA depletion on Ebp1-enhanced rRNA synthesis. Jurkat T cells were co-transfected with GFP-Ebp1 and SCR or siTIF-IA (20 nM) for 36 hours. 5′ETS pre-rRNA and RNA labeling (left) and Pol I binding and western blot (right). (F) Effects of Ebp1 depletion on rRNA synthesis and Pol I binding to rDNA in MEF cells. RNA and protein were extracted from MEF–Ebp1+/+ or MEF–Ebp1−/− cells. 5′ETS pre-rRNA levels and RNA labeling (left), ChIP assay with Pol I antibody (middle), and TIF-IA was immunoprecipitated and western blot probed with anti-Pol I antibody (right). (G) Comparison of effects of TIF-IA overexpression in MEF–Ebp1+/+ and MEF–Ebp1−/− cells. Cells were transfected with vector control or TIF-IA for 24 hours. 5′ETS pre-rRNA levels and RNA labeling (left) and ChIP assay and western blot (right). (H) Effects of co-overexpression of Ebp1 and TIF-IA in MEF–Ebp1−/− cells. Cells were transfected with TIF-IA alone or co-transfected with TIF-IA and Ebp1 for 24 hours. 5′ETS pre-rRNA levels and RNA labeling (left) and ChIP assay and western blot (right).

Enhancement of rRNA synthesis by co-expression of TIF-IA with Ebp1. (A) Effects of Ebp1 depletion on rRNA synthesis in T cells. Cultured T cells were transfected with SCR or siEbp1 (20 nM) for 36 hours. 5′ETS pre-rRNA levels and RNA labeling (left) and ChIP assay with Pol I antibody and western blot (right). (B) Effects of Ebp1 depletion on TIF-IA localization; 293T cells were transfected with SCR or siEbp1 (20 nM) for 36 hours. Cells were co-stained with anti–TIF-IA and anti-UBF antibodies (left). rDNA was labeled with a rDNA probe as described in “Methods.” Fluorescence intensity was measured along the line through 3D pictures on the left (right). (C) Effects of Ebp1 depletion on TIF-IA binding with Pol I in primary T cells. Cells were transfected with SCR or siEbp1 (20 nM) for 36 hours. Nucleoli were isolated (see “Methods”) and nucleolar or whole cell lysates were immunoblotted for TIF-IA, Ebp1, and NPM1 (left), whereas cell lysate was incubated with anti–TIF-IA and the precipitate immunoblotted with anti-Pol I antibody (right). (D) Effects of co-overexpression of TIF-IA and Ebp1 on rRNA synthesis. Jurkat T cells were co-transfected with GFP-Ebp1 and vector control or Myc–TIF-IA for 24 hours. 5′ETS pre-rRNA and RNA labeling (left) and Pol I binding by ChIP assay and western blot (right). (E) Effects of TIF-IA depletion on Ebp1-enhanced rRNA synthesis. Jurkat T cells were co-transfected with GFP-Ebp1 and SCR or siTIF-IA (20 nM) for 36 hours. 5′ETS pre-rRNA and RNA labeling (left) and Pol I binding and western blot (right). (F) Effects of Ebp1 depletion on rRNA synthesis and Pol I binding to rDNA in MEF cells. RNA and protein were extracted from MEF–Ebp1+/+ or MEF–Ebp1−/− cells. 5′ETS pre-rRNA levels and RNA labeling (left), ChIP assay with Pol I antibody (middle), and TIF-IA was immunoprecipitated and western blot probed with anti-Pol I antibody (right). (G) Comparison of effects of TIF-IA overexpression in MEF–Ebp1+/+ and MEF–Ebp1−/− cells. Cells were transfected with vector control or TIF-IA for 24 hours. 5′ETS pre-rRNA levels and RNA labeling (left) and ChIP assay and western blot (right). (H) Effects of co-overexpression of Ebp1 and TIF-IA in MEF–Ebp1−/− cells. Cells were transfected with TIF-IA alone or co-transfected with TIF-IA and Ebp1 for 24 hours. 5′ETS pre-rRNA levels and RNA labeling (left) and ChIP assay and western blot (right).

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