Fig. 5. Misexpression of CBF1^AA mutant proteins alters expression of SOHo1, GH6, EphA3, CBF2 and ephrin A2, but not ephrin A5. (A) Schematic representation of myc-tagged CBF1^AA mutant. The CBF1^AA mutant is deficient for DNA-binding activity because of substitutions of asparagine 189 and histidine 193 with alanines. (B) DNA pull-down assays using the nuclear extracts prepared from chick embryonic fibroblasts transfected with myc-tagged CBF1 or CBF1^AA (a). Western blot analysis using anti-myc primary antibody indicated the amounts of nuclear extracts used in the DNA pull-down assays (b). (C) Whole-mount in situ hybridization of E3 (stage 18-20) chick embryos transfected with CBF1^AA/RCAS using antisense probes for SOHo1 (a), GH6 (b), EphA3 (c) or CBF2 (d). The normal expression of SOHo1, GH6, EphA3 or CBF2 in the control eyes, is shown in insets. Arrowheads indicate the border of the endogenous expression. (D) Horizontal section in situ hybridization of E8 retina transfected with CBF1^AA/RCAS using antisense probes for EphA3 (a,b), ephrin A5 (c,d) or ephrin A2 (e,f). The temporal regions of untransfected retinae (control) are shown in the left panels (a,c,e), and those of the transfected ones in the same embryos are shown in the right panels (b,d,f). Scale bar: 100 µm.