MLL processing is not required for MLL-dependent gene activation.

<p>A. Schematic structures of MLL proteins produced by <i>dC</i> and <i>uc</i> mutations. The characteristics of each mutant are shown on the right. The mutated processing sites are highlighted in red. B. Sequences at the processing sites of the PCR fragments amplified from genomic DNAs of the recombined ES cell clones. C. Expression of MLL proteins in embryos. Western blotting was performed on whole embryo extracts of various genotypes. MLL proteins were visualized by anti-MLL^C antibody. Anti-menin blot serves as a loading control. <i>uc/+</i>: <i>uc</i> heterozygous. <i>uc/uc</i>: <i>uc</i> homozygous. <i>dC/+</i>: <i>dC</i> heterozygous. <i>dC/ dC</i>: <i>dC</i> homozygous. D. Genotypes at various developmental stages. Viability of embryos was confirmed by presence of heart contractions. E. Expression of <i>Hoxc8</i> transcripts in E10.5 embryos. Whole mount in situ hybridization was performed using the <i>Hoxc8</i> probe <i>(Hoxc8</i>). Arrows indicate sites of target gene expression. Previously published data for the wild type and <i>dC</i> homozygous embryos <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0073649#pone.0073649-Yokoyama1" target="_blank">[8]</a> are shown here for comparison. F. Expression of various genes in mutant MEFs. Three independently established MEF lines of wild typ<i>e</i>, <i>uc</i> homozygous and <i>dC</i> homozygous genotypes were examined by RT-qPCR for genes indicated at the tops of respective panels. Relative expression levels (normalized to <i>Gapdh</i>) are expressed relative to those of wild type-1 arbitrarily set as 1. Previously published data for wild type and <i>dC</i> homozygous MEFs <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0073649#pone.0073649-Yokoyama1" target="_blank">[8]</a> obtained in the same experiment are included for comparison. Error bars represent the standard deviations of triplicate PCRs. G. Proliferative capacities of <i>uc</i> homozygous MEFs. Proliferation assay (top) and 3T3 senescence assay (bottom) were performed for three lines each of wild type, <i>uc</i> homozygous and <i>dC</i> homozygous genotypes at passage 3. Previously published data for wild type and <i>dC</i> homozygous MEFs <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0073649#pone.0073649-Yokoyama1" target="_blank">[8]</a> obtained in the same experiments are included for comparison.