Inducible expression of DL1ICD variants in ES cells.

<p>(A) Schematic representations of the expression construct prior to and after Cre-mediated recombination, and of the pMP8 targeting vector. Black and white triangles indicate wild type loxP and mutant loxP2272 sites, respectively. (B) Western blot analysis of HA-tagged DΔECD expressed in CHO cells showing in addition to a γ-secretase-dependent cleavage product (arrow) a γ-secretase-independent proteolytic fragment (arrow head). (C) GFP expression in targeted ES cells indicating Cre-mediated activation of transgene expression. (D) Western blot analysis of cell lysates from wild type and DL1ICD-expressing E14tg2a cells using affinity purified polyclonal anti-DICD peptide antibodies.</p

Inefficient nuclear translocation and cleavage of DICD.

<p>(A) Detection of DICD stably (c, d) or transiently (e, f) expressed in CHO cells in the absence (c, e) or presence (d, f) of the proteasome inhibitor MG132. (B) Schematic representation of DICD-LexAVP16 fusion constructs. (C) Activation of lexA operator driven luciferase in CHO cells. (D) Western blot of cell lysates of CHO cells stably expressing DICD and fDICD.</p

Normal development of embryos expressing DL1ICD variants.

<p>(A) GFP expression in male, and hetero-and homozygous female transgenic embryos indicating Cre-mediated activation of transgene expression. (B) Western blot analysis of cell lysates from wild type and DL1ICD-expressing embryos. (C) Whole mount in situ hybridization of wild type (a, h) and DL1ICD-expressing (b-g, and i-n) embryos showing normal anterior-posterior somite patterning (a-g) and muscle differentiation (h-n). (D) Whole mount in situ hybridization of wild type (a) and DL1ICD-expressing (b-g) embryos showing normal neuronal differentiation. (E) qRT-PCR analysis of Nfem and Isl1 expression in wild type and DL1ICD-expressing embryos. Indicated are means and SEM of expression levels determined in individual wild type and transgenic embryos. ns: not significant (p>0.05).</p

Normal Notch target gene expression in embryos expressing DL1ICD variants.

<p>(A) Whole mount in situ hybridization of wild type and DL1ICD-expressing embryos showing normal Hey1 expression. (B) qRT-PCR analysis of Hes5 and Hey2 expression in wild type and DL1ICD-expressing embryos. Indicated are means and SEM of expression levels determined in individual wild type and transgenic embryos. ns: not significant (p>0.05).</p

Normal proliferation and neuronal differentiation of ES cells expressing DL1ICD variants.

<p>(A) Doubling times of targeted E14tg2a cells before and after Cre-mediated activation of DL1ICD expression. Doubling times were calculated from cell counts after non-linear regression using Prism software (GraphPad). Indicated are mean doubling times and upper and lower limit of 95% confidence intervals. (B) Western blot analysis of cell lysates of wild type and DL1ICD-expressing ES cells, CHO cells with or without transient expression of mouse p21, and HeLa nuclear extract. The arrow points to the position of p21, the asterisk marks a non-specific background band detected in ES cells. (C) Expression of the pan-neuronal marker Nefm in differentiated wild type and DL1ICD-expressing ES cells analyzed by qRT-PCR. Indicated are means and SEM of expression levels determined in differentiated wild type (n=16 pools of aggregates ) RA treated (n=14 pools of aggregates) and transgenic (DICD: n=13 pools of aggregates; fDICD: n=12 pools of aggregates; DΔECD: n=10 pools of aggregates) ES cells. ns: not significant (p>0.05). </p

Additional file 7 of Activity of the mouse Notch ligand DLL1 is sensitive to C-terminal tagging in vivo

Additional file 7: Figure S3. Overlay of bright field and chemoluminescence photographs of the Western blot membranes used for Fig. 1Ba-c. a-c correspond to a-c in Fig. 1B

Additional file 6 of Activity of the mouse Notch ligand DLL1 is sensitive to C-terminal tagging in vivo

Additional file 6: Table S3. Proteins detected in DLL1 complexes. Listed are significantly detected proteins. The full mass spectrometry data are available in the PRIDE database under Accession number PXD024680

Additional file 3 of Activity of the mouse Notch ligand DLL1 is sensitive to C-terminal tagging in vivo

Additional file 3: Figure S2. Surface biotinylation of tagged DLL1 proteins. (A) Western blots of cell lysates (input) and biotinylated proteins purified by Avidin beads (Avpd) from CHO cells expressing DLL1AcGFPHA. (a) Photograph of bound antibodies detected by chemoluminescence, (b) overlay of bright field and chemoluminescence photographs of Western blot membranes. Two aliquots from each of the 8 samples analysed per cell line (x.1 and x.2) were quantified relating the input to the Avpd band. Dotted lines indicate where membranes were cut. Primary antibodies used are indicated to the right. (B) Western blots of cell lysates (input) and biotinylated proteins purified by Avidin beads (Avpd) from CHO cells expressing DLL1SF. (a) Photograph of bound antibodies detected by chemoluminescence, (b) overlay of bright field and chemoluminescence photographs of Western blot membranes. Two aliquots from each of the 8 samples analyzed per cell line (x.1 and x.2) were quantified relating the input to the Avpd band. Dotted lines indicate where membranes were cut. Primary antibodies used are indicated to the right. (C) Western blots of cell lysates (input) and biotinylated proteins purified by Avidin beads (Avpd) or immunoprecipitated with anti-GFP (IP GFP) or anti-Flag (IP Flag) antibodies from CHO cells expressing DLL1AcGFPHA (left) or DLL1SF (right). (a) Photograph of bound antibodies detected by chemoluminescence, (b) overlay of bright field and chemoluminescence photographs of Western blot membranes. Dotted lines indicate where membranes were cut. Primary antibodies used are indicated to the right. DLL1Flag: lysate of CHO cells expressing flag-tagged DLL1 serving as positive control. Arrows point to biotinylated DLL1 purified by Avidin beads that is not immunoprecipitated by anti-GFP or anti-Flag antibodies. Asterisks indicate Ig heavy chains of primary antibodies used for immunoprecipitations detected by the secondary antibodies

A Novel Mammal-Specific Three Partite Enhancer Element Regulates Node and Notochord-Specific <em>Noto</em> Expression

<div><p>The vertebrate organizer and notochord have conserved, essential functions for embryonic development and patterning. The restricted expression of developmental regulators in these tissues is directed by specific cis-regulatory modules (CRMs) whose sequence conservation varies considerably. Some CRMs have been conserved throughout vertebrates and likely represent ancestral regulatory networks, while others have diverged beyond recognition but still function over a wide evolutionary range. Here we identify and characterize a mammalian-specific CRM required for node and notochord specific (NNC) expression of NOTO, a transcription factor essential for node morphogenesis, nodal cilia movement and establishment of laterality in mouse. A 523 bp enhancer region (NOCE) upstream the <em>Noto</em> promoter was necessary and sufficient for NNC expression from the endogenous <em>Noto</em> locus. Three subregions in NOCE together mediated full activity in vivo. Binding sites for known transcription factors in NOCE were functional in vitro but dispensable for NOCE activity in vivo. A FOXA2 site in combination with a novel motif was necessary for NOCE activity in vivo. Strikingly, syntenic regions in non-mammalian vertebrates showed no recognizable sequence similarities. In contrast to its activity in mouse NOCE did not drive NNC expression in transgenic fish. NOCE represents a novel, mammal-specific CRM required for the highly restricted <em>Noto</em> expression in the node and nascent notochord and thus regulates normal node development and function.</p> </div

DLL3 physically interacts with LFNG and is modified by POFUT and LFNG at the predicted O-fucosylation consensus motifs in EGF2 and EGF5.

<p>(A) Detection of DLL3 and LFNG interaction by coimmunoprecipitations of Flag-tagged DLL3 and HA-tagged LFNG expressed in CHO cells (red arrowheads point to co-precipitated proteins). (B) Schematic overview of expression constructs used for the analysis of DLL3 modifications. DLL3 carries a consensus motif for O-fucosylation (C²XXGG(S/T)C³) in EGF2 (S at position 286) and EGF5 (T at position 403). T83* and T206* are amino acids in the N-terminal and DSL region predicted to be modified by GalNAc O-glycosylation (NetOGlyc3.1 Server: <a href="http://www.cbs.dtu.dk/services//NetOGlyc" target="_blank">www.cbs.dtu.dk/services//NetOGlyc</a>, [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0123776#pone.0123776.ref047" target="_blank">47</a>]. Sig, Signal sequence; DSL, DSL domain; 1–6, EGF-like repeats 1 to 6; TM, transmembrane region; F, Flag tag, S286A, Serin mutation into Alanin; T403V, Threonin mutation into Valin. (C) Immunoprecipitations of wild type DLL3, DLL3-S286A, DLL3-T403V and DLL3-S286A,T403V proteins from lysates of metabolically labeled CHO cells stably expressing the respective protein. No tritiated fucose was incorporated into the DLL3-S286A,T403V protein (red arrowhead, lower row), indicating that these are the only O-fucosylation sites in DLL3. As a positive control for the metabolic labeling procedure cells were labeled with S³⁵-Methionine (middle row). Western blot analysis (upper row) with anti-Flag antibody shows different expression levels of used clones, consistent with different signal intensities after labeling with methionine. (D) Schematic representation of the strategy to analyze O-fucose elongation by LFNG using click-iT chemistry. LFNG catalyses elongation of O-linked fucose (black square) bound to Serin or Threonin in EGF-like repeats with N-Acetylglucosamine (grey circle) followed by Galactose (grey triangle) and Sialic Acid (white diamond) [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0123776#pone.0123776.ref045" target="_blank">45</a>]. With the "click" reaction azide modified Sialic acid is chemoselectively ligated to alkyne-tagged Biotin (white circle), which can be detected with Streptavidin or anti-Biotin Antibodies. (E and F) Immunoblots of Flag-tagged DLL3 variants shown in (B) immunoprecipitated from lysates of metabolically Ac₄ManNAz (sialic acid precursor) labeled CHO cells using anti-Flag antibodies. Incorporation of sialic acid (see D) was visualized with peroxidase-conjugated Streptavidin (E) or with anti-Biotin Antibody (F). Presence of immunoprecipitated DLL3 variant proteins was verified using anti-DLL3 antibodies (E) or on input Lysate with anti-Flag antibodies (F). DLL3 and the O-fucosylation mutant DLL3-S286A, T403V showed incorporation of sialic acid (E, red arrowheads), indicating the presence of additional O-Glycosylation sites. Sialic acid was incorporated into wild type DLL3 lacking the N-terminus and DSL domain (F, upper row, red arrowhead) but not into the truncated variant when S286 and T403 were mutated (DLL3ΔN,DSL-S286A, T403V, see B) indicating further modification of O-fucose residues at S283 and T403.</p