Fig. 5. Characterization of the ICER antibody.a, b, ICER immunoreaction could be abolished by preincubation of the antibody with the antigenic peptide in a dose-dependent manner in immunoblots (a) (2.5- to 250-fold excess of ICER protein) and immunohistochemical preparations (b) (250-fold excess of ICER protein). Similar results were obtained in two additional experiments. Scale bar, 20 μm. c, Gel mobility shift analyses with nuclear extracts obtained from rat pineal glands incubated with a labeled AA-NAT CRE always revealed a specific band of retarded mobility (lower arrow) that comigrated with bacterially generated ICER-IIγ (compare lanes 1, 4 with lanes 2, 3, 7–12). An additional ICER-specific signal of unknown quality is indicated by the upper arrow. Coincubation with the ICER antibody generated an additional low mobility complex (lanes 2, 3; indicated by a star). Excess of unlabeled AA-NAT CRE (100×) suppressed specific binding (comparelanes 5, 6 with lanes 7, 8). Notably, the labeled AA-NAT CRE (lanes 9–12) has a higher affinity for nuclear extracts as compared with that of a labeled somatostatin-CRE (lanes 13–16) (lanes 9, 11, 13, 15, 1 μg of nuclear extract; lanes 10, 12, 14, 16, 10 μg of nuclear extract). Similar results were obtained from four separate nuclear extract preparations.