Figure 3

C-terminal regions and ZFD of ZIC3 are required for trans-activation. (A, B) Schematic representation of wildtype ZIC3 and (A) PTC-containing mutants or (B) ZF mutants containing a missense mutation in the second cysteine (C) [converted to serine (S)]. (C–F) HEK293T cells were co-transfected with the reporter construct (above line) and expression constructs (below line) shown. For each transfection, luminescence was measured 24 h post-transfection in each of three replicate samples and each transfection repeated three times (N = 3). In each panel, the top graph (C–F) shows one representative experiment with the corresponding western blot. Error bars represent SD between the three replicates. Expression of the transfected proteins was confirmed with α-V5 and the α-LaminB1 (C, D) or α-TBP (E, F) blots served as nuclear fraction loading control. Although cropped blots were used, the gels were run under the same experimental conditions. The bottom graph (C′–F′) shows mean RLA (normalised to V5-DEST such that the V5-DEST value becomes 1), N = 3. Error bars represent SEM. a, b and c: p < 0.01 ANOVA.