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

Additional file 8: Figure S4. Overlay of bright field and chemoluminescence photographs of the Western blot membranes used for Fig. 2D a-c. a-c correspond to a-c in Fig. 2D

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

Additional file 4: Table S1. Quantification of DLL1AcGFPHA 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)

Additional file 2 of Activity of the mouse Notch ligand DLL1 is sensitive to C-terminal tagging in vivo

Additional file 2: Figure S1. Targeting of Dll1. (A) Targeting scheme (for details see Material and Methods). (B) Southern blots of BamHI-digested genomic ES cell DNA with radioactively labelled probes from the 5’ flank (a), the 3’flank (b), and puro (c). Left lanes at each panel show ethidium bromide-stained lanes from agarose gels. Correctly targeted clones are indicated at the top. wt = wild type ES DNA

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)

Additional file 1 of Activity of the mouse Notch ligand DLL1 is sensitive to C-terminal tagging in vivo

Additional file 1: Text S1. Detailed description of materials, primers, antibodies and methods

Lgr6 labels a rare population of mammary gland progenitor cells that are able to originate luminal mammary tumours

The mammary gland is composed of a complex cellular hierarchy with unusual postnatal plasticity. The identities of stem/progenitor cell populations, as well as tumour-initiating cells that give rise to breast cancer, are incompletely understood. Here we show that Lgr6 marks rare populations of cells in both basal and luminal mammary gland compartments in mice. Lineage tracing analysis showed that Lgr6+ cells are unipotent progenitors, which expand clonally during puberty but diminish in adulthood. In pregnancy or following stimulation with ovarian hormones, adult Lgr6+ cells regained proliferative potency and their progeny formed alveoli over repeated pregnancies. Oncogenic mutations in Lgr6+ cells resulted in expansion of luminal cells, culminating in mammary gland tumours. Conversely, depletion of Lgr6+ cells in the MMTV-PyMT model of mammary tumorigenesis significantly impaired tumour growth. Thus, Lgr6 marks mammary gland progenitor cells that can initiate tumours, and cells of luminal breast tumours required for efficient tumour maintenance