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

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

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 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

Additional file 5 of Activity of the mouse Notch ligand DLL1 is sensitive to C-terminal tagging in vivo

Additional file 5: Table S2. Quantification of DLL1SF cell surface presentation. The determined values of inputs (full length and cleaved product) were multiplied with the factor 50 and the Avidin pull downs (full length and cleaved product) with the factor 2,2 to calculate the total amount of detected DLL1 in the lysate and IP in each sample. Each sample was analysed twice (#x.1 and #x.2)

Somite patterning and vertebral column defects in single and double <i>Dll3</i> and <i>Lfng</i> mutants.

<p>Whole-mount in situ hybridizations of E9.5 embryos with an <i>Uncx4.1</i> specific probe (a-d) and lateral (e-h) and dorsal (e‘-h‘) views of skeletal preparations of wild type (a, e,e‘), homozygous <i>Dll3</i>^<i>pu</i> (b, f, f‘), homozygous <i>Lfng</i>^<i>lacZ</i> (c, g, g‘) and double homozygous <i>Dll3</i>^<i>pu</i>; <i>Lfng</i>^<i>lacZ</i> E18.5 embryos (d, h, h‘). Absence of both proteins does not enhance somite A-P patterning or vertebral column defects present in single mutants.</p