Journal: Oncogene

Article Title: Cyclin E and Bcl-xL cooperatively induce cell cycle progression in c-Rel-/- B cells.

doi: 10.1038/sj.onc.1206917

Figure Lengend Snippet: Figure 6 c-Rel regulates E2F3a expression in B cells. (a) Resting splenic B cells from c-rel þ / þ; bcl-xLTg (WT Tg) and c-rel/; bcl- xLTg (KO Tg) B cells were cultured for 36 h in the presence of 10 mg/ml anti-IgM and assayed by RT–PCR using primers specific for E2F3a. (b) Approximately 1 kb of mouse e2f3a promoter sequence is shown with two putative NF-kB/Rel-binding sites indicated by red boxes, as determined by Transfac database analysis. Site I corresponds to 864 bp upstream of the ATG translational start site ( þ 1) and site II is located 214 bp upstream. (c) Cells were cultured as described in (b) and assayed for NF-kB/Rel-binding activity by gel-shift assay using a radi- olabeled probe containing the 864 kB site and antibodies specific for p50, p65, and c-Rel to supershift complexes. The inducible complex (labeled A) is supershifted by p50, c-Rel, and p65 antibodies in wild-type B cells stimulated with 10 mg/ml anti-IgM, but a mutated site (864 kBMUT) prevents the binding of these complexes. (Supershifted complexes do not visibly migrate, but appear lighter than nonshifted control bands instead.) An additional site (214 kB) does not appear to bind any NF-kB/ Rel complexes either. Complex B represents nonspecific binding activity that serves to show equal loading of lanes. (d) CH31 B cells stimulated with 10 mg/ml anti-CD40 also exhibit c-Rel and p50 binding activity (labeled A) at the 864 kB site of the e2f3a promoter but a mutant 864 kB site and the 214 kB site do not bind. (e) WEHI 231 B cells stimulated with 10 mg/ml anti-CD40 show NF-kB/Rel binding activity (labeled A) while the mutant 864 kB site and 214 kB site do not exhibit any NF-kB/Rel- binding activity. (f) A total of 1 105 NIH 3T3 cells were transfected with 0.5 mg of firefly luciferase constructs containing a 2 kb e2f3a promoter (E2F3a-luc), a mutant 2 kb promoter (MUT- luc) bearing a mutation at the 864 kB site, or a positive control Igk chain promoter (Igk-luc), and cotransfected with either 0.5 mg of p50, c-Rel, or empty expression vector (pcDNA3). Cells were also cotransfected with a Renilla luciferase internal control plasmid containing a thymidine kinase promoter (tk-pRL) to drive constitutive Renilla luciferase activity for normalization of data. At 48 h, cells were harvested and assayed for firefly and Renilla luciferase activity. All experiments were carried out in triplicate and repeated in three separate experiments. A representative set of data is shown

Article Snippet: Supershift analysis was performed using 5–7mg anti-p130 (sc-317 ), anti-p107 (sc-317 ), anti-cRel (sc-071) Santa Cruz Biotechnology, anti-p50 from previous studies (Liou et al., 1994), and anti-p65 (RelA; 100–4165) from Rockland.

Techniques: Expressing, Cell Culture, Reverse Transcription Polymerase Chain Reaction, Sequencing, Binding Assay, Activity Assay, Gel Shift, Labeling, Control, Mutagenesis, Transfection, Luciferase, Construct, Positive Control, Plasmid Preparation