Fig. 3. Neither subcellular localization nor DNA binding is affected by mutations in the self-inhibitory domain of Bcd. (A) Western blot results for wild-type Bcd and Bcd(A52-56) in nuclear (N) and cytoplasmic (C) fractions of the transfected cells. Lanes 1 and 2 represent results from cells that were transfected with an empty effector plasmid expressing no Bcd. (B) DNA-binding assay using Bcd proteins synthesized in an in vitro transcription/translation system. The left panel shows the proteins (labeled with ³⁵S) and the right panel shows the gel shift results using a ³²P-labeled DNA probe containing a Bcd binding site. In both panels, lanes 1 to 3 represent no Bcd, wild-type Bcd and Bcd(A52-56), respectively. The Bcd-DNA complex is marked with a solid arrowhead. (C) Gel shift assays for a Scatchard analysis to determine the dissociation constants (K_D) for wild type Bcd (left panel) and Bcd(A52-56) (right panel) expressed in S2 cells. In this assay, the nuclear extracts generated from transfected S2 cells were used in gel shift assays with increasing concentrations of the radioactively labeled DNA probe: 5x10^–10 M, 1x10^–9 M, 2x10^–9 M, 5x10^–9 M, 1x10^–8 M and 3.3x10^–8 M for lanes 1 to 6, respectively. The solid arrowhead indicates the full-length Bcd-DNA complex, which was not formed using nuclear extracts made from non-transfected cells (not shown); smaller bands seen on the gel are presumably complexes containing breakdown products of Bcd. Quantitation was based on the amount of the full-length Bcd-DNA complex. (D) Scatchard plots for wild-type Bcd and Bcd(A52-56) expressed in S2 cells. Three independent assays yielded an estimated K_D value (–1/K_D=slope) of 3.0±0.9 nM and 4.0±0.5 nM for wild-type Bcd and Bcd(A52-56), respectively.