Xenbase Image ID: 146759

Figure 2. Human PRDM12 mutations cause structural instability and impair induction of sensory neuronal markers. (A) The structural modeling of the human PRDM12 mutations is based on the crystal structure of the human PRDM12 methyltransferase domain (PDB:3EP0). Mutated residues and the substitutions are colored in green and gray, respectively, and shown in stick representation. Protein is shown using cartoon and bonds representations. N- and C-terminal parts are colored in violet and cyan, respectively. (B) Western blot analysis using anti-flag antibodies to detect DDK-tagged wild type PRDM12 or the indicated human PRDM12 mutant proteins transiently transfected into HEK cells. Note that we failed to detect the truncation mutant S58fs. (C) Immunofluorescence analysis of HEK cells transiently transfected with DDK-tagged human wild type PRDM12 (WT) or the human R160C, D31Y, and E172D PRDM12 mutants. Arrows indicate protein aggregates of the mutant R160C and E172D protein as well as loss of nuclear staining for the mutant D31Y. The DDK-tag was visualized with anti-flag antibodies (green); nuclei are counterstained with DAPI (blue). Overlays appear in white. Representative images are shown. Magnifications 100×. (D) Real-time RT-PCR analysis of the expression of the indicated genes (±SEM) in stage 26 Xenopus animal cap explants isolated from embryos injected with noggin mRNA, together with wild type or mutated human Prdm12 mRNAs as indicated and treated with retinoic acid (RA). Data are presented as the mean ± SEM of 3 idependent experiments. Expression levels (fold change) were normalized to GAPDH and compared to the expression level of noggin-injected RA treated caps, which was defined as 1. Significance was determined by Student's t-test, where p-values were defined as: * P ≤ 0.05; **P ≤ 0.01; *** P ≤ 0.001. Image published in: Nagy V et al. (2015) Copyright © 2015. This image is reproduced with permission of the publisher and the copyright holder. This is an Open Access article distributed under the terms of the Creative Commons Attribution License. Larger Image Printer Friendly View

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