Figure 7
Electrophoretic mobility-shift assays with NEs with MEFs from WT and Jdp2KO mice. (a) NEs from Jdp2KO MEFs (indicated as K), WT MEFs (indicated as W) and bovine serum albumin (BSA; indicated as B were incubated with probes corresponding to the CRE (lanes 1–3), AP1-like sequence (AP1L; lanes 4 and 5), the AP-1 point mutant (mAP1, lanes 6 and 7) and AP-1 (lanes 8–16) in the presence of 1 and 2 pmol of unlabeled mAP1 oligodeoxynucleotide (lanes 12 and 13) or AP-1 oligodeoxynucleotide (lanes 10 and 11) as a competitor. The positions of shifted bands (B1–B4) and free DNA probes (Free) are indicated. (b) Super-shifting electrophoretic mobility-shift assay using NEs from serum-starved MEFs (FCS−) or 15% serum-induced MEFs (FCS+) and the DNA probe that corresponded to AP-1. NEs from WT MEFs (W) and Jdp2KO MEFs (K) were incubated without (odd lanes) or with (even lanes) antibodies specific for JDP2. ‘*’ indicates the super-shifted bands and X indicates aggregated DNA–protein complexes that remained in the wells of the gel. B1–B4 and Free indicate protein–DNA complexes and free DNA probes. (c) Competition and super-shifting assays with the AP-1 DNA probe. Reaction mixtures containing NEs from WT MEFs (lanes 1–9) and Jdp2KO MEFs (lanes 10–18) were prepared with antibodies specific for c-Jun, JunB, JunD, c-Fos, ATF-2, E2F1 and JDP2 (lanes 1–18), as indicated. ‘*’ indicates super-shifted bands of antibody–DNA–protein complexes. B1–B4 and Free indicate DNA–protein complexes and the unbound AP-1 probe. (d) Competition and super-shifting assays using NEs of WT MEFs and the CRE DNA probe. Reaction mixtures containing NEs from WT MEFs were prepared without (lane 1) and with 0.1, 1 and 10 pmol of unlabeled (cold) CRE probe (lanes 2–4) and antibodies specific for JDP2, pJDP2, c-Jun, JunB, JunD, c-Fos and ATF-2 (lanes 5–11), as indicated. B2 indicates super-shifted bands of antibody–DNA–protein complexes. B1 and Free indicate DNA–protein complexes and the unbound CRE probe. AP, activation protein; FCS, fetal calf serum; JDP2, Jun dimerization protein-2; KO, Jdp2-knockout; MEF, murine embryonic fibroblast; NE, nuclear extract; WT, wild type.
