FIG. 4.

NRSE binding activity in PC12 and A126.1B2 cell lines. (A) Electrophoretic mobility shift assay. Nuclear cell extracts prepared from the indicated cell lines were analyzed for binding to the cholinergic NRSE/RE-1 by electrophoretic mobility shift assays. (B) Western blot analysis of NRSF. Nuclear extracts (100 μg) from each cell line were subjected to SDS-PAGE followed by Western blotting with an anti-NRSF MAb and detection with the ECL+Plus detection system. PC12 cells were treated with 10 μM H89 as for Fig. 2. (C) Supershifting of NRSE/RE-1 gel shift band. Electrophoretic mobility shift assays were conducted as described for panel B except that the nuclear extract was preincubated with 2 μg of anti-NRSF MAb or 2 μg of a control MAb prepared against mouse ChAT where indicated.