Figure 3

Antiserum against unique region of OSBZ8 factor recognizes OSBZ8 from the ABRE-EMSA complex by either causing supershift or in western analysis after elution of the EMSA complex from the dried gel. A. The nuclear extracts from IR-72 (lane 1) and Pokkali (lane 2 to 5) were incubated with the purified antibody before and after the addition of the probe. Super shift was only detected in post-incubation of the antiserum (lane 1, 2 and 5) and not in pre-incubation (lane 4). In lane 3, antiserum was not added. B. Western blot analysis of protein extracted from dried gel with shifted complex shown in fig. 3A. The nuclear factor was eluted from the [³²P]ABRE: protein complex or [³²P]ABRE: protein: Ab complex from dried native polyacrylamide gel. It was analyzed by silver staining (IR-72 in Lane 1; Pokkali in Lane 2 and Pokkali-Ab complex in Lane 3, Panel I); recognition by the antiserum against N-terminal end of OSBZ8 (38-kD) factor (Panel II) but not by the preimmune serum (Panel III) after SDS-PAGE.