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. 2007 Feb 12;27(8):2861–2869. doi: 10.1128/MCB.02276-06

FIG. 2.

FIG. 2.

hRev7 activates Elk-1-dependent transcription. (A to E) Luciferase reporter assays of the indicated GAL4 fusion proteins in the presence of hRev7 on a GAL4-driven TK promoter-reporter plasmid (0.25 μg) (shown schematically at the top of panels A and B). (A) Dose-dependent Elk-1 activation. Constructs encoding GAL4-Elk-1(223-428) (0.2 μg) were transfected in the presence of increasing amounts of hRev7 constructs (0, 0.5 μg, 1.5 μg) in 293T cells. Where indicated, cells were treated with 0.01% MMS (+). Rel, relative; α-GAL4 DBD, anti-GAL4 DBD antibody. (B and C) Specificity of hRev7 effect. Each indicated GAL4-TAD fusion construct (0.1 μg) was transfected into 293T cells in the absence (−) and presence (+) of hRev7 (1 μg) (B) or increasing amounts (0.2 and 1 μg) of hRev7 or hMAD2 constructs (C). IB, immunoblotting. (D and E) Activity of Elk-1 following hRev7 knockdown. (D) GAL4-Elk-1(223-428) activity was monitored in control 293 cells or two different 293 cells stably expressing shRNA constructs against hRev7 in the presence (+) and absence (−) of MMS stimulation. Data are presented as changes in induction by MMS. (E) Rescue of Elk-1 activity by hRev7 reexpression. GAL4-Elk-1(223-428) activity was monitored in control and hRev7 knockdown cells in the presence and absence of MMS treatment and/or cotransfection of hRev7 containing a silent mutation (hRev7_sil). All luciferase reporter data were performed in duplicate, and the averages from three independent experiments are shown. Western blots are shown in the panels below each experiment with immunoblotting with the indicated antibodies to show the expression levels of transfected and endogenous proteins. (F) Luciferase reporter assays of the indicated egr-1 promoter-reporter plasmid (0.1 μg) (shown schematically at the top) in the presence of Elk-1 (25 ng) and increasing amounts of hRev7 constructs (0, 0.1 μg, 1 μg, and 2 μg) in 293T cells. Rel. Luc Activity, relative luciferase activity. (G) Quantitative RT-PCR analysis of egr-1, c-fos, and β-actin expression in MMS-treated HeLa cells. Endogenous hRev7 protein was reduced by siRNA treatment, and siRNA duplexes against GAPDH (siGAPDH) were used as a negative control. Experiments were performed in duplicate, and data were averaged from two independent experiments. Rel, relative.