Fig 2: Immunohistochemistry of ZBED6 and ZC3H11A in brain and muscle tissue from mouse and in pig muscle.(A–J) Immunofluorescence analysis of mouse tissues stained with anti-ZBED6 or anti-ZC3H11A antibodies. (A and D) Coronal brain overview images assembled from pictures taken at 4× magnification. (B, C, E, and F) Higher magnification images of boxed areas indicated in (A and D) with anti-ZBED6 (B and C) or anti-ZC3H11A (E, F) immunopositive signals in red. (G–J) Images of anti-ZBED6 or anti-ZC3H11A antibody stainings of section from mouse thigh muscle. Nuclear localization of ZBED6 and ZC3H11A is confirmed by colabeling with DAPI (blue) in both brain and muscle as indicated by arrowheads, whereas nuclei lacking ZBED6- or ZC3H11A-positive signals are indicated by arrows. (K–N) Immunofluorescence analysis of pig muscle tissue (longissimus dorsi) stained with anti-ZBED6 (K and L) or anti-ZC3H11A (M and N). α-Bungarotoxin fused to a fluorescent dye (αBTX, green) was used to confirm identification of skeletal muscle tissue since this toxin binds cholinergic receptors in neuromuscular junctions. Scale bars indicate 480 µm in (A and D), 22 µm in (B, C, E, and F), and 14 µm in (G–N).

Fig 3: Identification of ZBED6.(A) Mass spectrometric quantification of ZBED6-enrichment using SILAC; the core sequences of the wild-type q and the mutant Q oligonucleotides used for protein capture are shown on top. ZBED6 was identified by six unique peptides in the SILAC-heavy q sample. Two peptides were simultaneously observed in the SILAC-light Q sample and were used as the basis for SILAC quantification. A representative peptide is shown where the area under the curve corresponds to the amount of ZBED6 enriched by the q probe (blue) and the Q probe (red). The average overall enrichment of ZBED6 by q was 9.0-fold (±1.2-fold, n = 2 experiments). (B) Genome organization of Zbed6 and Zc3h11a in mouse, adapted from the UCSC Genome Browser (http://genome-test.cse.ucsc.edu/). Untranslated and translated exons of Zc3h11a are indicated by black and red bars, respectively. A mammalian conservation track from the UCSC browser is included at the bottom. (C) Schematic representation of the six ZBED family members present in human and the Hermes hAT DNA transposase from the house fly. (D) Phylogenetic analysis of ZBED protein sequences from selected species. Cf (Canis familiaris; dog), Gg (Gallus gallus; chicken), Hs (Homo sapiens; human), Md (Monodelphis domestica; opossum), Mm (Mus musculus; house mouse), and Oa (Ornithorhynchus anitinus; platypus). ZBED1 and ZBED2 homologs were not found in the mouse genome, and mouse Zbed5 contains numerous mutations and stop codons efficiently destroying the reading frame. (E) EMSA showing binding of recombinant ZBED6 BED1/2 domains to wild-type (q), but not to mutant (Q) probe (left panel). Likewise, endogenous ZBED6 in nuclear extracts (NE) from C2C12 cells forms a complex with wild-type probe that is competed by q, but not Q probe (right panel). The complex is supershifted by the anti-ZBED6 antibody, and competition confirmed the specific interaction with ZBED6.