Figure 6.
A Point Mutation, Y25F or Y25H, Disrupting c-Rel's DNA-Binding Ability Compromises Its Repressive Function on RelA-Dependent Transactivation
(A) Amino acid sequence alignment of N-terminal DNA-binding regions of c-Rel and v-Rel, with critical residues for DNA binding highlighted.
(B) HEK 293T cells (3 × 105) were transfected in a six-well plate with plasmids encoding FLAG-tagged RelA and WT or Y25F c-Rel together with luciferase reporter plasmids as indicated in the figure.
(C) HEK 293T cells (3 × 105) were transfected in a six-well plate with FLAG-tagged Y25F c-Rel and the indicated luciferase reporter plasmid. Eighteen hours following transfection, cells were stimulated with 100 ng/mL TNF-α for 6 h. Luciferase activity was assessed using a dual luciferase assay system. Data are presented as mean ± standard error of mean (SEM). p Values were obtained by unpaired Student’s t test; ***p < 0.001, **p < 0.01, ns non significant. (B and C) Data in bar graphs are technical triplicates representative of three independent experiments (n = 3). RelA alone value in Figure 6B was from the same representative experiment in Figure 2A for accurate comparison. Bottom: Western blotting analysis of total cell lysates of luciferase assay using anti-FLAG-tag antibody. Blots are representative of three independent experiments.
(D) c-Rel knockout MEFs (3 × 106) were transfected with FLAG-tagged wild-type or Y25F c-Rel. Eighteen hours following transfection, MEFs were left untreated or treated with 100 ng/mL TNF-α for 15 min. Nuclear lysates equivalent to 200 μg of nuclear proteins per sample were utilized for in vitro pull-down assay using the Ig-κB oligonucleotide. Nuclear lysates were probed with indicated antibodies. hnRNPA1 was used as loading control.
(E) HEK 293T cells (2 × 106/10-cm plate) were transfected with FLAG-tagged wild-type or Y25F c-Rel. Cells were stimulated and analyzed as in (D).
(F) Cells were transfected with c-Rel Y25H, and experiments were performed as in (B).
(G) Cells were transfected with c-Rel Y25H, and experiments were performed as in (C).
(H) HEK 293T cells were transfected with FLAG-tagged wild-type or Y25H c-Rel as in (E). Cells were stimulated and analyzed as in (D).
Data for (D), (E), and (H) are representative of three independent experiments. See also Figure S5.