Fig. 3. The M1 domain is sufficient to confer Meis activity. (A) All constructs used are expressed at comparable levels in embryos. One- to two-cell stage embryos were injected with 300 pg of each mRNA encoding Myc-tagged constructs as indicated at the top of each lane. Embryos were raised to 5 hpf, lysed, resolved on a 10% SDS-PAGE gel, western blotted and probed with anti-Myc antibody. (B-M) Analysis of Pbx4/Lzr-mediated nuclear localization of Meis constructs. One- to two-cell stage embryos were injected with 300 pg of each mRNA as indicated at the bottom right of each panel, raised to 5 hpf and stained with anti-Myc antibody. All Meis constructs were Myc-tagged, whereas Pbx4/Lzr was untagged. (N-U) Analysis of in vivo activity of Meis constructs. One- to two-cell stage embryos were injected with 500 pg lacZ RNA (control) or 165 pg of each mRNA as indicated in the lower right corner of each panel, raised to 25 hpf and analyzed for expression of hoxb1a (N-Q) or hoxb2 (R-U) by in situ hybridization. All embryos are dorsal views with anterior to the left. (V) Meis3-Pbx4 fusion constructs do not bind endogenous Pbx. One- to two-cell stage embryos were injected with 300 pg MycMeis3 (lane 1) or MycBMNPbx4 (lane 2) and raised to 10 hpf. Embryos were lysed, immunoprecipitated with anti-Myc, resolved on a 10% SDS-PAGE gel, western blotted and probed with anti-Pbx4 antiserum (left panel) or anti-Myc antiserum (right panel). Note that the BMNPbx4 fusion protein in lane 2 of the left-hand panel is detected by the anti-Pbx4 antiserum. MycMeis3 and BMNPbx4 are the same size. IgH, antibody heavy chain; IgL, antibody light chain. (W) Meis3-Pbx4 fusion proteins remain stable at 12 hpf. One- to two-cell stage embryos were injected with 300 pg MycBMNPbx4 or MycIPbx4 mRNA and harvested at 5 hpf or 12 hpf. Embryos were lysed, and three embryo equivalents were resolved on a 10% SDS-PAGE gel, western blotted and probed with anti-Myc antiserum.