Journal: Respiratory physiology & neurobiology

Article Title: Paralemmin-3 augments lipopolysaccharide-induced acute lung injury with M1 macrophage polarization via the notch signaling pathway.

doi: 10.1016/j.resp.2023.104203

Figure Lengend Snippet: Fig. 5. Effect of PALM3 expression on Notch signaling pathway in AMs isolated from the BALF of each group. (A) Notch signaling associated molecules (NICD, RBP-J and IRAK2) mRNA and protein expression in in AMs isolated from the BALF of each group. (B) Co-immunoprecipitation assays of NICD and IRAK2 with RBP-J in AMs isolated from the BALF of each group. All data are expressed as means ± SD (n = 6). *P < 0.05 vs. normal group, and #P < 0.05 vs. empty vector group.

Article Snippet: Immunoprecipitation was performed with anti-rat-NICD antibody (1:50, #4147 monoclonal rabbit anti-rat, Cell Signaling Technology, Inc., Danvers, MA, USA), anti-ratRBP-J antibody (1:100, sc-271128, Santa Cruz) or normal rat IgG (1:100, sc-2026, Santa Cruz).

Techniques: Expressing, Isolation, Immunoprecipitation, Plasmid Preparation

Journal: Respiratory physiology & neurobiology

Article Title: Paralemmin-3 augments lipopolysaccharide-induced acute lung injury with M1 macrophage polarization via the notch signaling pathway.

doi: 10.1016/j.resp.2023.104203

Figure Lengend Snippet: Fig. 6. Involvement of Notch signaling pathway in PALM3-mediated M1 polarization. (A) The frequency of F4/80+CD16+ cells in AMs isolated from BALF were determined by flow cytometry with PE and FITC staining. All data are expressed as means ± SD (n = 6). *P < 0.05 vs. lenti.r-PALM3 + DMSO group, and #P < 0.05 vs. NS + DMSO group. (B) The mRNA levels of M1 specific genes (TNF-α, IL-1, CCr7 and iNOS) in AMs isolated from BALF were determined by qRT-PCR. All data are expressed as means ± SD (n = 6). *P < 0.05 vs. NS + DMSO group, and #P < 0.05 vs. lenti.r-PALM3 + DMSO group. (C) The mRNA levels of Notch signaling associated molecules (NICD, RBP-J and IRAK2) in AMs isolated from BALF were determined by qRT-PCR. All data are expressed as means ± SD (n = 6). *P < 0.05 vs. NS + DMSO group, and #P < 0.05 vs. lenti.r-PALM3 + DMSO group. (D) Co-immunoprecipitation assays of NICD and IRAK2 with RBP-J in AMs isolated from BALF. All data are expressed as means ± SD (n = 6). *P < 0.05 vs. NS + DMSO group, and #P < 0.05 vs. lenti.r-PALM3 + DMSO group.

Article Snippet: Immunoprecipitation was performed with anti-rat-NICD antibody (1:50, #4147 monoclonal rabbit anti-rat, Cell Signaling Technology, Inc., Danvers, MA, USA), anti-ratRBP-J antibody (1:100, sc-271128, Santa Cruz) or normal rat IgG (1:100, sc-2026, Santa Cruz).

Techniques: Isolation, Flow Cytometry, Staining, Quantitative RT-PCR, Immunoprecipitation

Journal: Respiratory physiology & neurobiology

Article Title: Paralemmin-3 augments lipopolysaccharide-induced acute lung injury with M1 macrophage polarization via the notch signaling pathway.

doi: 10.1016/j.resp.2023.104203

Figure Lengend Snippet: Fig. 7. Co-immunoprecipitation assays of NICD and RBP-J with PALM3 in AMs isolated from BALF. After rats were pretreated with lentiviral vectors, the AMs were collected from BALF on day 7 after successive LPS inhalations. The total cell lysates were immunoprecipitated with anti-NICD, anti-RBP-J or normal IgG antibodies. Then, the immunocomplexes were examined by western blot analysis using the indicated antibodies. All data are expressed as means ± SD (n = 6). *P < 0.05 vs. normal rat group.

Article Snippet: Immunoprecipitation was performed with anti-rat-NICD antibody (1:50, #4147 monoclonal rabbit anti-rat, Cell Signaling Technology, Inc., Danvers, MA, USA), anti-ratRBP-J antibody (1:100, sc-271128, Santa Cruz) or normal rat IgG (1:100, sc-2026, Santa Cruz).

Techniques: Immunoprecipitation, Isolation, Western Blot

Journal:

Article Title: Notch alters VEGF responsiveness in human and murine endothelial cells by direct regulation of VEGFR-3 expression

doi: 10.1172/JCI24311

Figure Lengend Snippet: (A) Diagram of Notch protein structure. N1IC encodes the cytoplasmic domain of Notch1. N4/int-3 encodes 30 amino acids upstream of the transmembrane domain, the transmembrane domain, and the cytoplasmic domain of Notch4 fused to a HA tag. (B) Western blot analysis of Ad-LacZ or N4/int-3 endothelial cells using anti-HA antibody. (C) Activation of a CSL luciferase reporter in endothelial cells expressing N4/int-3, represented as fold induction in Ad–N4/int-3 cells relative to Ad-LacZ. Data are an average of 3 independent experiments. (D) qRT-PCR of Tie and VEGFRs in Ad-infected endothelial cells. Values were normalized to β-actin qRT-PCR. Data of 3 independent qRT-PCR analyses are represented as fold induction in Ad–N4/int-3 cells relative to Ad-LacZ. (E) Western blot analysis of cell surface proteins isolated from Ad-LacZ and Ad–N4/int-3 endothelial cells immunoblotted with anti–VEGFR-3 antibody and total cell lysates immunoblotted with anti–α-tubulin antibody. (F) Western blot analysis of cell surface proteins (CSPs) isolated from Ad-LacZ–, Ad–N4/int-3–, and Ad-N1IC–infected endothelial cells immunoblotted with antibodies against VEGFR-3 and α-tubulin. Total cell lysates (TCLs) immunoblotted with antibodies against the HA epitope of N4/int-3 (12CA5), N1IC, and α-tubulin. (G and H) VEGFR-3, Hey1, and Hey2 qRT-PCR of Jagged1/Notch4 (J1/N4) or Dll4/Notch4 (Dll4/N4) HUVEC cocultures, respectively. Values were normalized by β-actin qRT-PCR and are presented as relative values. (I) VEGFR-2 and VEGFR-3 FACS of Ad-LacZ, Ad-N1IC, and Ad–N4/int-3 BECs.

Article Snippet: Human IMCs and murine ovary and dorsal skin sections (5 μm) immunostained with antibodies against PECAM (Pharmingen), Notch4 (J. Kitajewski laboratory or Santa Cruz Biotechnology Inc.), VEGFR-3 (eBioscience or R&D Systems), LYVE-1 (R&D Systems), Podoplanin (Angiobio), or Cleaved Notch1 (Val1744) Antibody (Cell Signaling Tech).

Techniques: Western Blot, Activation Assay, Luciferase, Expressing, Quantitative RT-PCR, Infection, Isolation

Journal:

Article Title: Notch alters VEGF responsiveness in human and murine endothelial cells by direct regulation of VEGFR-3 expression

doi: 10.1172/JCI24311

Figure Lengend Snippet: Viability of E9.5 and P21 mice from Notch1+/– and VEGFR-3+/– crosses

Article Snippet: Human IMCs and murine ovary and dorsal skin sections (5 μm) immunostained with antibodies against PECAM (Pharmingen), Notch4 (J. Kitajewski laboratory or Santa Cruz Biotechnology Inc.), VEGFR-3 (eBioscience or R&D Systems), LYVE-1 (R&D Systems), Podoplanin (Angiobio), or Cleaved Notch1 (Val1744) Antibody (Cell Signaling Tech).

Techniques:

Journal:

Article Title: Notch alters VEGF responsiveness in human and murine endothelial cells by direct regulation of VEGFR-3 expression

doi: 10.1172/JCI24311

Figure Lengend Snippet: Notch1+/– (A and D), VEGFR-3+/– (B and E), and Notch1+/–;VEGFR-3+/– (C and F) E9.5 embryos were immunostained for PECAM. (D–F) The dorsal region of embryos was enlarged to visualize the dorsal aorta (blue arrowheads), atria of the primary heart tube (green arrowheads), and intersomitic vessels. E9.5 VEGFR-3+/– (G and I) and Notch1+/–;VEGFR-3+/– (H and J) embryos were β-gal stained to visualize the VEGFR-3–positive vasculature. (I and J) The cranial region of the embryos was enlarged to demonstrate the decrease in VEGFR-3 expression (red asterisks) and reduction of VEGFR-3–positive vessel density (yellow arrowheads). Original magnification, ×5 (A–C, G, and H); ×15 (D–F); ×10 (I and J).

Article Snippet: Human IMCs and murine ovary and dorsal skin sections (5 μm) immunostained with antibodies against PECAM (Pharmingen), Notch4 (J. Kitajewski laboratory or Santa Cruz Biotechnology Inc.), VEGFR-3 (eBioscience or R&D Systems), LYVE-1 (R&D Systems), Podoplanin (Angiobio), or Cleaved Notch1 (Val1744) Antibody (Cell Signaling Tech).

Techniques: Staining, Expressing

Journal:

Article Title: Notch alters VEGF responsiveness in human and murine endothelial cells by direct regulation of VEGFR-3 expression

doi: 10.1172/JCI24311

Figure Lengend Snippet: Murine P4 dermal sections immunostained with PECAM (A and J), VEGFR-3 (D and M), LYVE-1 (G and P), Notch1 (B, E, and H), or Notch4 (K, N, and Q) antibodies. Notch1 is coexpressed with PECAM (C), VEGFR-3 (F), and LYVE-1 (I). Notch4 is coexpressed with PECAM (L), VEGFR-3 (O), and LYVE-1 (R). (M–R) White arrowheads highlight Notch4 staining in the dermal lymphatic endothelium. Original magnification, ×12.5 (A–C and J–L); ×40 (D–I and M–R).

Article Snippet: Human IMCs and murine ovary and dorsal skin sections (5 μm) immunostained with antibodies against PECAM (Pharmingen), Notch4 (J. Kitajewski laboratory or Santa Cruz Biotechnology Inc.), VEGFR-3 (eBioscience or R&D Systems), LYVE-1 (R&D Systems), Podoplanin (Angiobio), or Cleaved Notch1 (Val1744) Antibody (Cell Signaling Tech).

Techniques: Staining

Journal:

Article Title: Notch alters VEGF responsiveness in human and murine endothelial cells by direct regulation of VEGFR-3 expression

doi: 10.1172/JCI24311

Figure Lengend Snippet: Human IMC immunostained with VEGFR-3 (B and E), LYVE-1 (H and L), podoplanin (P), Notch1 (A and G), Notch4 (D and K), or activated Notch1 (N1Val) (O) antibodies. Notch1 (C) and Notch4 (F) are coexpressed with VEGFR-3 in the extratumoral IMC vessels. Notch1 (I) and Notch4 (M) are coexpressed with LYVE-1 in the extratumoral lymphatics. J and N represent magnified view of boxed areas in I and M, showing lymphatic vessels. (N) White arrowhead indicates Notch4-positive LEC. Yellow arrowheads highlight Notch4-positive non-endothelial cells adjacent to the lymphatic endothelium. (Q) The activated Notch1 peptide is localized to the LEC nuclei. White arrowheads highlight positive nuclei, and yellow arrowhead indicates a negative nucleus. Original magnification, ×50 (A–F and O–Q); ×40 (G–I and K–M); ×120 (J and N).

Article Snippet: Human IMCs and murine ovary and dorsal skin sections (5 μm) immunostained with antibodies against PECAM (Pharmingen), Notch4 (J. Kitajewski laboratory or Santa Cruz Biotechnology Inc.), VEGFR-3 (eBioscience or R&D Systems), LYVE-1 (R&D Systems), Podoplanin (Angiobio), or Cleaved Notch1 (Val1744) Antibody (Cell Signaling Tech).

Techniques:

Journal: Development (Cambridge, England)

Article Title: NOTCH3 signalling controls human trophoblast stem cell expansion and differentiation.

doi: 10.1242/dev.202152

Figure Lengend Snippet: Fig. 1. Expression and localization of NOTCH receptors, ligands and MAML co-activators in purified trophoblast subtypes and first trimester placental tissues. (A,B) Relative mRNA expression in matched CTBs and STBs, purified by a two-step protease digestion protocol from first trimester placentae (n=7, 6th to 12th week), was measured by RT-qPCR (duplicates). Data were normalized to transcript levels of TATA box-binding protein (TBP). Data are mean±s.e.m. *P<0.05, **P<0.01, ***P<0.001 (unpaired, two-tailed Student’s t-test). ns, not significant; nd, not detectable; AU, arbitrary units. (C,D) Representative western blots showing protein expression in the isolated trophoblast samples of early placentae (n=3, 7th to 9th week). GAPDH was used as a loading control. (E) In situ localization in first trimester placental tissues. Representative immunofluorescence images of placental sections from 6th (n=3) and 12th week (n=3) of gestation are shown. Dashed lines indicate the border between villous cytotrophoblast (CTB) and syncytiotrophoblast (STB). Higher magnifications of the outlined areas are shown in the insets. Scale bars: 50 µm. TEAD4 and CDH1 (E-cadherin) mark the CTB cell layer. Nuclei are stained with DAPI. Images with a 10 µm scale bar show selected CTB nuclei expressing NOTCH3-ICD. VC, villous core.

Article Snippet: Immunoprecipitations were performed using NOTCH3 or MAML1 antibodies, as well as appropriate rabbit IgG controls (listed in Table S1) according to manufacturer’s instructions (Cell Signaling, 73778).

Techniques: Expressing, Purification, Quantitative RT-PCR, Binding Assay, Two Tailed Test, Western Blot, Isolation, Control, In Situ, Immunofluorescence, Staining

Journal: Development (Cambridge, England)

Article Title: NOTCH3 signalling controls human trophoblast stem cell expansion and differentiation.

doi: 10.1242/dev.202152

Figure Lengend Snippet: Fig. 2. Expression and localization of NOTCH receptors, ligands and MAML co-activators in self-renewing and differentiated trophoblast stem cells. (A,B) RT-qPCR analyses measuring transcript levels in proliferating TSCs (−cAMP; n=6) and differentiated TSCs, treated with forskolin for 5 days (+cAMP, n=6). Data were normalized to TBP (AU, arbitrary units). Data are mean±s.e.m. *P<0.05, **P<0.01, ***P<0.001 (unpaired, two-tailed Student’s t-test). ns, not significant; nd, not detectable. (C,D) Representative western blots showing protein expression in proliferating and cAMP-differentiated TSCs (n=3). TEAD4/YAP1 and ENDOU were selected as markers of stemness and cell fusion, respectively. GAPDH was used as loading control. (E) Immunofluorescence in self-renewing and cAMP-treated TSCs (n=3). Scale bars: 25 µm. CDH1 (E-cadherin) and SDC1 were used as markers of expanding and fused TSCs, respectively. (F) Immunoprecipitation in cell lysates, prepared from proliferating TSCs (n=3), using antibodies binding NOTCH3 and MAML1. A representative example is shown. IgG was used as negative control. IP, immunoprecipitation.

Article Snippet: Immunoprecipitations were performed using NOTCH3 or MAML1 antibodies, as well as appropriate rabbit IgG controls (listed in Table S1) according to manufacturer’s instructions (Cell Signaling, 73778).

Techniques: Expressing, Quantitative RT-PCR, Two Tailed Test, Western Blot, Control, Immunofluorescence, Immunoprecipitation, Binding Assay, Negative Control

Journal: Development (Cambridge, England)

Article Title: NOTCH3 signalling controls human trophoblast stem cell expansion and differentiation.

doi: 10.1242/dev.202152

Figure Lengend Snippet: Fig. 6. NOTCH3-ICD promotes trophoblast expansion and impairs differentiation. (A,D) Representative western blots detecting protein expression of trophoblast stemness- and proliferation/mitosis-associated genes upon overexpression of NOTCH3-ICD in (A) primary CTBs (n=3) and (D) TSCs (n=3). GAPDH was used as loading control. (B) Representative immunofluorescence images showing EdU labelling in TSCs that express NOTCH3-ICD. Scale bars: 50 µm. Percentage of EdU+ TSCs (n=9) was measured by counting EdU+/DAPI ratio of 10 individual areas per experiment and condition, each containing between 800 and 950 nuclei. Data are mean±s.e.m. ***P<0.001 (unpaired, two-tailed Student’s t-test). Arrowheads indicate EdU+ nuclei. (C) Immunofluorescence images illustrating SDC1+ areas (outlined) in the absence or presence of exogenous NOTCH3-ICD. A representative experiment is shown. Scale bars: 200 µm. To determine the size of SDC1+ areas, 10 different regions of NOTCH3-ICD-expressing TSCs (n=9) and controls, each containing between 650 and 800 nuclei, were evaluated. Data are mean±s.e.m. ***P<0.001 (unpaired, two-tailed Student’s t-test).

Article Snippet: Immunoprecipitations were performed using NOTCH3 or MAML1 antibodies, as well as appropriate rabbit IgG controls (listed in Table S1) according to manufacturer’s instructions (Cell Signaling, 73778).

Techniques: Western Blot, Expressing, Over Expression, Control, Immunofluorescence, Two Tailed Test

Journal: Development (Cambridge, England)

Article Title: NOTCH3 signalling controls human trophoblast stem cell expansion and differentiation.

doi: 10.1242/dev.202152

Figure Lengend Snippet: Fig. 7. Schematic depiction of the role of canonical NOTCH3 signalling in human trophoblast cells. Active NOTCH3-ICD, interacting with the transcriptional co-activator MAML1, promotes self-renewal of CTB progenitors and TSCs, and maintains its expression in an autocrine manner, whereas downregulation of the NOTCH3 pathway results in STB formation.

Article Snippet: Immunoprecipitations were performed using NOTCH3 or MAML1 antibodies, as well as appropriate rabbit IgG controls (listed in Table S1) according to manufacturer’s instructions (Cell Signaling, 73778).

Techniques: Expressing

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: The Hippo pathway effector TAZ induces intrahepatic cholangiocarcinoma in mice and is ubiquitously activated in the human disease

doi: 10.1186/s13046-022-02394-2

Figure Lengend Snippet: Primary antibodies used for immunohistochemistry (IHC) and Western blot analysis (WB)

Article Snippet: Notch2 , IHC , 1:50 , Cell Signaling Technology , 5732.

Techniques: Immunohistochemistry, Western Blot, Concentration Assay

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: The Hippo pathway effector TAZ induces intrahepatic cholangiocarcinoma in mice and is ubiquitously activated in the human disease

doi: 10.1186/s13046-022-02394-2

Figure Lengend Snippet: Validated Gene Expression Assays used for real-time qRT-PCR experiments

Article Snippet: Notch2 , IHC , 1:50 , Cell Signaling Technology , 5732.

Techniques: Gene Expression

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: The Hippo pathway effector TAZ induces intrahepatic cholangiocarcinoma in mice and is ubiquitously activated in the human disease

doi: 10.1186/s13046-022-02394-2

Figure Lengend Snippet: Liver lesions developed in AKT/TAZ mice display activation of Hippo and AKT/mTOR pathways. ( A ) Representative immunohistochemical patterns of a cholangiocellular tumor exhibiting immunoreactivity for HA-Tag(AKT) and nuclear TAZ, as well as for downstream effectors of the Hippo (NOTCH1, NOTCH2, and JAG1) and AKT/mTOR (phosphorylated/activated AKT or p-AKT; phosphorylated/inactivated GSK-3β or p-GSK-3β; and phosphorylated/activated RPS6 or p-RPS6) pathways. Original magnification: 200x; scale bar: 100 μm. ( B ) Upregulation of the Hippo pathway targets CCN1 , CCN2 , and NOTCH 2 in AKT/TAZ livers (10 weeks post-injection) compared with livers injected with the empty vector (Vector), as assessed by quantitative real-time RT-PCR. Abbreviations: H&E, hematoxylin and eosin staining; ST, non-tumorous surrounding tissue; T, tumor. Data are expressed as means ± SD. *** p < 0.0001 and ** p < 0.01 vs. empty vector-injected mice

Article Snippet: Notch2 , IHC , 1:50 , Cell Signaling Technology , 5732.

Techniques: Activation Assay, Immunohistochemical staining, Injection, Plasmid Preparation, Quantitative RT-PCR, Staining

Journal: Cancer research

Article Title: EGFR blockade enriches for lung cancer stem-like cells through Notch3-dependent signaling

doi: 10.1158/0008-5472.CAN-13-3724

Figure Lengend Snippet: EGFR mediated negative regulation of Notch3 transcriptional activity in a tyrosine kinase-dependent manner. A–B, HEK293FT cells were transiently transfected with either 12X-CSL reporter (A) or full length HES1 reporter (B) with empty vector or N3DA construct and increasing amounts of wild type or kinase inactive EGFR to determine the role of EGFR on transcriptional activity of Notch3-ICD. The data presented are the average of three assays. C, Erlotinib stimulates gene expression of Hes1. HCC827 cells were treated with erlotinib for the indicated time periods and RNA was analyzed by qRT-PCR for the expression of Hes1.

Article Snippet: Antibodies Following antibodies were used in this study: EGFR (1005) Santa Cruz Biotechnology, EGFR (Ab12) and EGFR (Ab15) are from Neomarker, Notch1 (5B5), Notch3 (8G5), and Notch3 (D11B8) and EGFR (pY1173) obtained from Cell Signaling Technology.

Techniques: Activity Assay, Transfection, Plasmid Preparation, Construct, Gene Expression, Quantitative RT-PCR, Expressing

Journal: Cancer research

Article Title: EGFR blockade enriches for lung cancer stem-like cells through Notch3-dependent signaling

doi: 10.1158/0008-5472.CAN-13-3724

Figure Lengend Snippet: Depletion of Notch3 abolishes erlotinib-induced ALDH+ cells. A, HCC4006 cells were transfected with either non-targeting control (NTC-siRNA) (top) in the absence or presence of erlotinib as indicated, Notch1 siRNAs -1, -2 and -3 (middle), or Notch3 siRNAs -1, -2, and -3 (bottom). Forty-eight hours post-transfection, cells were treated with 0.1μM erlotinib for 3 days and ALDH activity was measured. As a control, NTC siRNA treated cells were also treated with DMSO (top, middle panel). A portion of the cells was pre-incubated with the ALDH inhibitor DEAB (+DEAB) to provide a gate for ALDH-negative cells (top left panel). B, HCC827 cells were transfected with non-targeting control pool (NTC-siRNA), pool of Notch3 or Notch1 siRNAs. Forty-eight hours following transfection, cells were treated with 0.1 μM erlotinib (B) for 3 days and ALDH activity was measured. As a control, NTC siRNA treated cells were also treated with DMSO (top, left panel). A similar experiment was also performed with an individual siRNA (bottom panel).

Article Snippet: Antibodies Following antibodies were used in this study: EGFR (1005) Santa Cruz Biotechnology, EGFR (Ab12) and EGFR (Ab15) are from Neomarker, Notch1 (5B5), Notch3 (8G5), and Notch3 (D11B8) and EGFR (pY1173) obtained from Cell Signaling Technology.

Techniques: Transfection, Control, Activity Assay, Incubation

Journal: Cancer research

Article Title: EGFR blockade enriches for lung cancer stem-like cells through Notch3-dependent signaling

doi: 10.1158/0008-5472.CAN-13-3724

Figure Lengend Snippet: EGF-mediated association between EGFR and Notch 3 receptors in HCC2429 cells is dependent on EGFR kinase activity. A. HCC2429 cells were stimulated with EGF (25 ng/ml) for the indicated times. Cell lysate was prepared from each time point and immunoprecipitated with EGFR antibody. Western analysis of precipitated proteins was performedand probed with a Notch3 antibody (top) and an EGFR antibody (middle). Whole cell lysate was subjected to western analyses using Notch3, and β-tubulin antibodies to detect the total expression of these proteins. B, HCC4006 C, HCC827cells were treated with DMSO or 0.1 μM erlotinib for 24 hours. Cell lysate was prepared from each treatment and immunoprecipitated with EGFR antibody and blotted for Notch3, or reciprocal immunoprecipitation was performed by precipitating Notch3 and blotting for EGFR. Whole cell lysate was subjected to western analyses using Notch3, EGFR and actin antibodies to detect the total expression of these proteins. IP= Immunoprecipitation, WCL = Whole Cell Lysate.

Article Snippet: Antibodies Following antibodies were used in this study: EGFR (1005) Santa Cruz Biotechnology, EGFR (Ab12) and EGFR (Ab15) are from Neomarker, Notch1 (5B5), Notch3 (8G5), and Notch3 (D11B8) and EGFR (pY1173) obtained from Cell Signaling Technology.

Techniques: Activity Assay, Immunoprecipitation, Western Blot, Expressing

Journal: Cancer research

Article Title: EGFR blockade enriches for lung cancer stem-like cells through Notch3-dependent signaling

doi: 10.1158/0008-5472.CAN-13-3724

Figure Lengend Snippet: EGF mediated tyrosine phosphorylation of the Notch3 receptor. A&B, HCC2429 cells were stimulated with EGF (25 ng/ml) for the indicated times to detect the ligand induced tyrosine phosphorylation of Notch3 receptor. A Cell lysates were immunoprecipitated with Notch3 antibody followed by western analysis using a phosphotyrosine antibody. The blot was stripped and re-probed with anti-Notch3 (bottom). B, A reciprocal immunoprecipitation was done with a phosphotyrosine antibody with a subsequent western blot probed for Notch3 (top). Equal amount of cell lysate was analyzed for Notch3 expression (middle) and tubulin (bottom).

Article Snippet: Antibodies Following antibodies were used in this study: EGFR (1005) Santa Cruz Biotechnology, EGFR (Ab12) and EGFR (Ab15) are from Neomarker, Notch1 (5B5), Notch3 (8G5), and Notch3 (D11B8) and EGFR (pY1173) obtained from Cell Signaling Technology.

Techniques: Phospho-proteomics, Immunoprecipitation, Western Blot, Expressing

Journal: Cancer research

Article Title: EGFR blockade enriches for lung cancer stem-like cells through Notch3-dependent signaling

doi: 10.1158/0008-5472.CAN-13-3724

Figure Lengend Snippet: EGFR kinase activity is necessary for the tyrosine phosphorylation of the Notch3 receptor. A, HCC4006 and B, HCC827 cells were treated with DMSO or erlotinib (0.1 μM) for indicated periods of time. Cell lysates from each time point were either precipitated with a phosphotyrosine antibody followed by a western blot probed with Notch3 antibody (top), or immunoprecipitated with Notch3 antibody followed by a western blot probed with a phosphotyrosine antibody. Whole cell lysate was also subjected to western analysis to check for of levels Notch3, EGFR-pY1173, and tubulin.

Article Snippet: Antibodies Following antibodies were used in this study: EGFR (1005) Santa Cruz Biotechnology, EGFR (Ab12) and EGFR (Ab15) are from Neomarker, Notch1 (5B5), Notch3 (8G5), and Notch3 (D11B8) and EGFR (pY1173) obtained from Cell Signaling Technology.

Techniques: Activity Assay, Phospho-proteomics, Western Blot, Immunoprecipitation

Journal: Physiological reports

Article Title: Long noncoding RNA TINCR is a novel regulator of human bronchial epithelial cell differentiation state.

doi: 10.14814/phy2.14727

Figure Lengend Snippet: FIGURE 4 (a) Selected gene expressions regarding epithelial mesenchymal markers, differentiation makers and regulators from RNA- sequencing data. (b and c) qRT–PCR and western blot analysis of SOX2, HES1, JAG1, NOTCH1 and NOTCH2 expression at day4 after siRNA. *p < 0.05. Data presented are from one of two independent experiments with similar results. (d and e) qRT–PCR and western blot analysis of SOX2, HES1, JAG1, NOTCH1 and NOTCH2 expression for NHBECs transfected with pcDNA3.1-EGFP-Blank or pcDNA3.1-EGFP-TINCR. *p < 0.05. Data presented are from one of two independent experiments with similar results. (f) qRT–PCR analysis of SOX2, HES1, JAG1, NOTCH1 and NOTCH2 expression at day3 and day14 after airlift with siRNA and 3D organoid matrigel. *p < 0.05. Data presented are from one of two independent experiments with similar results. (g) qRT–PCR analysis of SOX2, HES1, JAG1, NOTCH1 and NOTCH2 expression at day3 and day14 after airlift with transfection of pcDNA3.1-EGFP-Blank or pcDNA3.1-EGFP-TINCR

Article Snippet: Rabbit anti-SOX2 antibody (cat. No 3579T), rabbit anti-HES1 antibody (cat. No 11988S), rabbit anti-JAG1 antibody (cat. No 2620T), rabbit anti-NOTCH1 antibody (cat. No 3608S), and rabbit anti-NOTCH2 antibody (cat. No 5732T) were from Cell Signaling Technology.

Techniques: RNA Sequencing, Quantitative RT-PCR, Western Blot, Expressing, Transfection

Journal: Physiological reports

Article Title: Long noncoding RNA TINCR is a novel regulator of human bronchial epithelial cell differentiation state.

doi: 10.14814/phy2.14727

Figure Lengend Snippet: FIGURE 5 TINCR binds to STAU1 protein and controls critical regulators of differentiation. (a) Bar graphs show percentage of TINCR in the cytoplasm (black) and nucleus (white). NEAT1 serves as a positive control for nucleus enriched RNA and GAPDH serves as a positive control for cytoplasmic RNA. Data presented are from two independent experiments. (b) RIP experiments were performed using isotype IgG and STAU1 antibody to immunoprecipitated STAU1 protein/mRNAs complexes in total-cell extracts of NHBECs, and relative enrichment was determined as RNA associated with STAU1 IP relative to an input control. Relative ARF1 enrichment served as a positive control and NEAT1 as a negative control as NEAT1 does not interact with STAU1. Data presented are from two independent experiments. (c) Relative mRNA enrichment of STAU1 antibody in total-cell extracts of NHBECs transfected with siSCR or siTICNR. Data presented are from two independent experiments. (d and e) qRT–PCR and western blot analysis of TINCR, TP63, SOX2, HES1, JAG1, NOTCH1 and NOTCH2 expression for NHBECs transfected with siSCR or siSTAU1. NHBECs were seeded on 6 well plate at 2 × 105 density and analyzed at day4 after transfection of siRNA reagents. *p < 0.05. (f and g) qRT–PCR and western blot analysis of TINCR, TP63, SOX2, HES1, JAG1, NOTCH1 and NOTCH2 expression for NHBECs transfected with siSCR or siSTAU after transfection of pcDNA3.1-EGFP-Blank or pcDNA3.1-EGFP-TINCR for 4 h. NHBECs were seeded on 24 well plate at 1 × 105 density and analyzed at day4 after transfection of siRNA reagents

Article Snippet: Rabbit anti-SOX2 antibody (cat. No 3579T), rabbit anti-HES1 antibody (cat. No 11988S), rabbit anti-JAG1 antibody (cat. No 2620T), rabbit anti-NOTCH1 antibody (cat. No 3608S), and rabbit anti-NOTCH2 antibody (cat. No 5732T) were from Cell Signaling Technology.

Techniques: Positive Control, Immunoprecipitation, Control, Negative Control, Transfection, Quantitative RT-PCR, Western Blot, Expressing

Journal: PLoS ONE

Article Title: Notch Activation Is Dispensable for D, L-Sulforaphane-Mediated Inhibition of Human Prostate Cancer Cell Migration

doi: 10.1371/journal.pone.0044957

Figure Lengend Snippet: Immunoblotting for cleaved and full-length Notch1, Notch2, and Notch4 using lysates from (A) PC-3, (B) LNCaP, and (C) LNCaP-C4-2B cells after 8-, 16-, or 24-hour treatment with DMSO or SFN (10 or 20 µM). Blots were stripped and re-probed with anti-actin antibody as a loading control. Immunoblotting for each protein was done at least twice using independently prepared lysates. Numbers above band represent changes in protein levels relative to corresponding DMSO-treated control.

Article Snippet: Antibodies against cleaved Notch1, and full-length Notch2 were from Cell Signaling Technology (Danvers, MA); an antibody specific for detection of cleaved Notch2 was from EMD-Millipore (Billerica, MA); antibodies against full-length Notch1, Notch4 (this antibody detects both full-length and cleaved forms), and HES-1 were from Santa Cruz Biotechnology (Santa Cruz, CA); and anti-actin antibody was from Sigma-Aldrich (St. Louis, MO).

Techniques: Western Blot

Journal: PLoS ONE

Article Title: Notch Activation Is Dispensable for D, L-Sulforaphane-Mediated Inhibition of Human Prostate Cancer Cell Migration

doi: 10.1371/journal.pone.0044957

Figure Lengend Snippet: (A) Chemical names, common names, and chemical formulae of analogs used in the present study. Immunoblotting for cleaved Notch1, Notch2, and Notch4 using lysates from (B) PC-3, and (C) LNCaP cells after 8-hour treatment with DMSO or different analogs (10 or 20 µM). Blots were stripped and re-probed with anti-actin antibody as a loading control. Immunoblotting for each protein was done at least twice using independently prepared lysates. Numbers above band represent changes in protein levels relative to DMSO-treated control.

Article Snippet: Antibodies against cleaved Notch1, and full-length Notch2 were from Cell Signaling Technology (Danvers, MA); an antibody specific for detection of cleaved Notch2 was from EMD-Millipore (Billerica, MA); antibodies against full-length Notch1, Notch4 (this antibody detects both full-length and cleaved forms), and HES-1 were from Santa Cruz Biotechnology (Santa Cruz, CA); and anti-actin antibody was from Sigma-Aldrich (St. Louis, MO).

Techniques: Western Blot

Journal: PLoS ONE

Article Title: Notch Activation Is Dispensable for D, L-Sulforaphane-Mediated Inhibition of Human Prostate Cancer Cell Migration

doi: 10.1371/journal.pone.0044957

Figure Lengend Snippet: (A) Immunoblotting for full-length Notch1 protein using lysates from PC-3 and LNCaP cells transiently transfected with a control (nonspecific) siRNA or a Notch1-targeted siRNA. (B) Representative images (Boyden chamber assay) depicting migration of PC-3 and LNCaP cells transfected with a control (nonspecific) siRNA or a Notch1-targeted siRNA and treated for 24 hours with DMSO or 10 μM SFN (×100 magnification). (C) Quantitation of PC-3 and LNCaP cell migration from data shown in panel B. Two to three fields on each filter were scored for cell migration under an inverted microscope. Data represent percent cell migration normalized to control siRNA-transfected cells treated with DMSO (mean ± SD, n = 6; combined data from two independent experiments each performed in triplicate). Significantly different ( P <0.05) a compared with respective DMSO-treated controls (cells transfected with control siRNA or Notch1-targeted siRNA), and b between control siRNA transfected cells and Notch1-targeted siRNA transfected cells by one-way ANOVA followed by Bonferroni's multiple comparison test. Each experiment was performed twice.

Article Snippet: Antibodies against cleaved Notch1, and full-length Notch2 were from Cell Signaling Technology (Danvers, MA); an antibody specific for detection of cleaved Notch2 was from EMD-Millipore (Billerica, MA); antibodies against full-length Notch1, Notch4 (this antibody detects both full-length and cleaved forms), and HES-1 were from Santa Cruz Biotechnology (Santa Cruz, CA); and anti-actin antibody was from Sigma-Aldrich (St. Louis, MO).

Techniques: Western Blot, Transfection, Boyden Chamber Assay, Migration, Quantitation Assay, Inverted Microscopy

Journal: PLoS ONE

Article Title: Notch Activation Is Dispensable for D, L-Sulforaphane-Mediated Inhibition of Human Prostate Cancer Cell Migration

doi: 10.1371/journal.pone.0044957

Figure Lengend Snippet: (A) Representative immunohistochemical images depicting expression of cleaved Notch1, cleaved Notch2, and HES-1 proteins in the prostatic intraepithelial neoplasia (PIN), well-differentiated prostate cancer (WD), and poorly-differentiated prostate cancer (PD) in the dorsolateral prostates from TRAMP mice of the indicated groups (×200 objective magnification). (B) Quantitation of expression (combined analysis in PIN, WD, and PD) is shown as H-score (mean ± SD; n = 5). Statistical significance was determined by Student's t -test.

Article Snippet: Antibodies against cleaved Notch1, and full-length Notch2 were from Cell Signaling Technology (Danvers, MA); an antibody specific for detection of cleaved Notch2 was from EMD-Millipore (Billerica, MA); antibodies against full-length Notch1, Notch4 (this antibody detects both full-length and cleaved forms), and HES-1 were from Santa Cruz Biotechnology (Santa Cruz, CA); and anti-actin antibody was from Sigma-Aldrich (St. Louis, MO).

Techniques: Immunohistochemical staining, Expressing, Quantitation Assay

Journal: Molecular cancer therapeutics

Article Title: Target deconvolution of a multikinase inhibitor with anti-metastatic properties identifies TAOK3 as a key contributor to a cancer stem cell-like phenotype

doi: 10.1158/1535-7163.MCT-18-1011

Figure Lengend Snippet: Organotypic 3D spheroids harbor enriched traits of cancer stem cells (CSCs). A. Light microscopy of PANC1 cells grown as 2D monolayer (left) and 3D spheroids (right) (40× magnification, scale bars indicate 20 µm). B. Gene expression measured by qRT-PCR in cell lines PANC1, L3.6pl, and KLM-1, bars plot ratio of mean CSC gene expression levels in cells grown as spheroids vs monolayer. Expression levels in monolayer cells was set to 1 and used as a reference to calculate the fold difference in spheroids (in triplicates; N=5 independent experiments, error bars indicate SEM). C. Phosphoproteomic profiling of PANC1 spheroids vs monolayer cells. Heat map derived from reverse protein microarrays (RPMA), color scale shows log-transformed values normalized to total protein. D. Immunofluorescence staining with anti-cleaved NOTCH1. Bar graph depicts mean intensity of 100 examined PANC1 cells (in triplicates; N=3 experiments), representative images shown on bottom (scale bars indicate 5 µm). E. Histograms of PANC1 cells grown as spheroids (red) and adherent monolayer cells (blue) stained with PE-conjugated mouse monoclonal antibody against human CD133 and normalized to histogram respective isotype controls (green; brown). F. Immunofluorescence staining of γ-H2AX in PANC1 spheroids and monolayer cells treated with 3 µM gemcitabine for 24 hours, and 24 hours after discontinuation of gemcitabine (100 cells examined, N=3 independent experiments). Representative images of γ-H2AX positive nuclei shown on bottom, caspase 3/7 levels of PANC1 monolayer cells and spheroids after treatment with 3 µM gemcitabine for 72 hours shown on right (levels set to 1 of vehicle-treated control). G. Tumor growth 8 weeks after subcutaneous injection of primary low-passage patient-derived SB.06 (left) and PANC1 (right) monolayer (2D) and spheroid cells (3D) into female nu/nu mice.

Article Snippet: Cells were permeabilized in 0.25% TritonX-100 and blocked with 5% normal goat serum in PBS at room temperature in a humidified chamber for 2 h. Slides were incubated with rabbit monoclonal anti-human NOTCH1 (Cat. #4380, Cell Signaling Technology), cleaved NOTCH1 (Cat. #4147, Cell Signaling Technology) and mouse monoclonal anti-human phospho-H2A.X (Cat. #05-636-AF555; Millipore Sigma, Burlington, MA) primary antibody, or rabbit IgG or mouse G3A1 IgG 1 isotype control overnight in 4°C.

Techniques: Light Microscopy, Gene Expression, Quantitative RT-PCR, Expressing, Derivative Assay, Transformation Assay, Immunofluorescence, Staining, Control, Injection

Journal: Molecular cancer therapeutics

Article Title: Target deconvolution of a multikinase inhibitor with anti-metastatic properties identifies TAOK3 as a key contributor to a cancer stem cell-like phenotype

doi: 10.1158/1535-7163.MCT-18-1011

Figure Lengend Snippet: Inhibitor #1 inhibits metastasis formation in vivo. A. Real-time monitoring of impact of inhibitor #1 on PANC1luc2 spheroid progression in orthotopic pancreatic cancer metastasis model. Tumor burden in excised distal organs in relation to primary pancreatic tumor quantified by bioluminescence imaging. Color bar indicates the intensity of the luminescent signal, with red and blue serving as the high and low signals (captured in triplicate determinations and presented as mean for each animal; N≥8 per group, representative images of excised organs are shown). B. Liver metastasis counted on H&E stains for both vehicle- and inhibitor #1-treated animals (right). Representative H&E stains of liver sections treated with vehicle and inhibitor #1 shown on left (scale bars indicate 100 µm). C. Inhibitor #1 decreases cleaved NOTCH1 expression in liver metastasis of treated NOD/IL2 gamma (null) PANC1 mice. Co-immunohistochemical staining of vehicle- and inhibitor #1-treated liver lesions (margins to uninvolved liver parenchym indicated by black line) with E-Cadherin (red chromophore) and cleaved NOTCH1 (visualized by brown chromophore). Percent positive cells within metastasis shown on right.

Article Snippet: Cells were permeabilized in 0.25% TritonX-100 and blocked with 5% normal goat serum in PBS at room temperature in a humidified chamber for 2 h. Slides were incubated with rabbit monoclonal anti-human NOTCH1 (Cat. #4380, Cell Signaling Technology), cleaved NOTCH1 (Cat. #4147, Cell Signaling Technology) and mouse monoclonal anti-human phospho-H2A.X (Cat. #05-636-AF555; Millipore Sigma, Burlington, MA) primary antibody, or rabbit IgG or mouse G3A1 IgG 1 isotype control overnight in 4°C.

Techniques: In Vivo, Imaging, Expressing, Immunohistochemical staining, Staining

Journal: Molecular cancer therapeutics

Article Title: Target deconvolution of a multikinase inhibitor with anti-metastatic properties identifies TAOK3 as a key contributor to a cancer stem cell-like phenotype

doi: 10.1158/1535-7163.MCT-18-1011

Figure Lengend Snippet: Loss of TAOK3 reduces cancer stem cell traits in pancreatic cancer cells. A. SOX2, NANOG, and CD44 mRNA expression measured by qRT-PCR (UnTx, untreated cells; Scramble KD, scramble siRNA). B. TAOK3 loss decreases anchorage-independent growth. Colony formation on soft agar 14 days after transfection with scramble and anti-TAOK3 siRNA (y-axis depicts number of colonies; N=3). C. Treatment with inhibitor #1 reduces colony formation compared to gemcitabine. Number of colonies of PANC1 monolayer cells and spheroids after treatment with 100 nM and 500 nM inhibitor #1 or 3 µM gemcitabine. Colony numbers after 14 days are depicted on y-axis. D. Expression levels of stemness genes measured by qRT-PCR after 24 hours of treatment with inhibitor #1 and gemcitabine compared to vehicle-treated values in both PANC1 monolayer cells and spheroids. E. Inhibitor #1 reduces cleaved NOTCH1 levels in PANC1 cells. Quantitative immunofluorescence of PANC1 2D monolayer cells (representative images, top row) and spheroids (bottom) treated for 24 hours with 500 nM inhibitor #1 or 3 µM gemcitabine. Quantification of mean fluorescence intensity (100 cells examined, N=2 independent experiments, in triplicates) on bottom. F. Inhibitor #1 reduces CD44 expression on PANC1 spheroids. Flow cytometry histogram of vehicle (blue) and inhibitor #1-treated (red) PANC1 spheroids.

Article Snippet: Cells were permeabilized in 0.25% TritonX-100 and blocked with 5% normal goat serum in PBS at room temperature in a humidified chamber for 2 h. Slides were incubated with rabbit monoclonal anti-human NOTCH1 (Cat. #4380, Cell Signaling Technology), cleaved NOTCH1 (Cat. #4147, Cell Signaling Technology) and mouse monoclonal anti-human phospho-H2A.X (Cat. #05-636-AF555; Millipore Sigma, Burlington, MA) primary antibody, or rabbit IgG or mouse G3A1 IgG 1 isotype control overnight in 4°C.

Techniques: Expressing, Quantitative RT-PCR, Transfection, Immunofluorescence, Fluorescence, Flow Cytometry

Journal: Nature Communications

Article Title: Plasma membrane-derived extracellular microvesicles mediate non-canonical intercellular NOTCH signaling

doi: 10.1038/s41467-017-00767-2

Figure Lengend Snippet: Enrichment of NOTCH pathway components in ARMMs

Article Snippet: Primary antibodies used in the study include rabbit polyclonal ARRDC1 antibody as described previously , mouse monoclonal CD9 antibody (Santa Cruz, catalogue #sc-13118, at 1:1000 dilution), rabbit monoclonal GFP antibody (Cell Signaling, catalogue #2956S, at 1:1000 dilution) and rabbit monoclonal NOTCH2 and ITCH antibodies (Cell Signaling, catalogue #4530S, 12117S, both at 1:2000 dilution).

Techniques:

Journal: Nature Communications

Article Title: Plasma membrane-derived extracellular microvesicles mediate non-canonical intercellular NOTCH signaling

doi: 10.1038/s41467-017-00767-2

Figure Lengend Snippet: NOTCH2 receptor is secreted into ARMMs. a Schematic drawing of domains in NOTCH2 receptor. Relative positions of peptides identified by mass spectrometry were indicated. ECD: extracellular domain; TM: transmembrane domain; NICD: NOTCH intracellular domain. b Western blotting of NOTCH2 protein in ARMMs. HEK293T cells were transfected with GFP or ARRDC1-GFP. Extracellular vesicles were pelleted by ultracentrifugation. Both cell lysates and extracellular vesicle ( EV ) pellets were used for anti-NOTCH2, anti-ARRDC1 and anti-CD9 western blotting. c Effect of ARRDC1 knockout on NOTCH release into ARMMs. CRISPR was used to knock out the ARRDC1 gene in HEK293T cells. EVs from conditioned culture media of ARRDC1-KO and wild-type HEK293T cells were pelleted and subjected to western blotting along with corresponding cell lysates. d Re-expression of ARRDC1 restored NOTCH2 release into ARMMs in ARRDC1-KO cells. ARRDC1-KO HEK293T cells were transfected with the indicated amount of ARRDC1-GFP construct. Two days after transfection, EVs were pelleted from the conditioned media and subjected to western blotting along with the cell lysates

Article Snippet: Primary antibodies used in the study include rabbit polyclonal ARRDC1 antibody as described previously , mouse monoclonal CD9 antibody (Santa Cruz, catalogue #sc-13118, at 1:1000 dilution), rabbit monoclonal GFP antibody (Cell Signaling, catalogue #2956S, at 1:1000 dilution) and rabbit monoclonal NOTCH2 and ITCH antibodies (Cell Signaling, catalogue #4530S, 12117S, both at 1:2000 dilution).

Techniques: Mass Spectrometry, Western Blot, Transfection, Knock-Out, CRISPR, Expressing, Construct

Journal: Nature Communications

Article Title: Plasma membrane-derived extracellular microvesicles mediate non-canonical intercellular NOTCH signaling

doi: 10.1038/s41467-017-00767-2

Figure Lengend Snippet: ITCH interacts with ARRDC1 and mediates NOTCH2 incorporation into ARMMs. a ARMMs contain ITCH. HEK293T cells were transfected with GFP or ARRDC1-GFP. EVs were pelleted by ultracentrifugation. Both cell lysates and EVs were used for anti-ITCH, anti-ARRDC1 and anti-CD9 western blotting. b Effect of ARRDC1 knockout on ITCH release into EVs. EVs from conditioned culture media of ARRDC1-KO and wild-type HEK293T cells were pelleted and subjected to anti-ITCH, anti-ARRDC1 and anti-CD9. c Co-immunoprecipitation ( IP ) showing the interaction between ARRDC1 and ITCH. HEK293T cells were co-transfected with Flag-tagged ITCH and one of the following: control vector, HA-tagged-ARRDC1 or ARRDC1-delPPXY (deletion of two PPXY motifs). Two days after transfection, cell lysates were collected and incubated with anti-HA agarose beads for immunoprecipitation. IP-ed complexes were subjected to anti-HA or anti-Flag western blotting. d Effect of ITCH knockdown on NOTCH2 release into ARMMs. HEK293T cells were co-transfected with ITCH siRNA (or scrambled control) and ARRDC1-GFP (or control GFP). Three days after transfection, EVs from conditioned culture media were pelleted and subjected to western blotting. The amount of NOTCH2 in EV was quantified. Data represent an average of three independent repeats. e Model depicting the role of ITCH on NOTCH2 release into ARMMs

Article Snippet: Primary antibodies used in the study include rabbit polyclonal ARRDC1 antibody as described previously , mouse monoclonal CD9 antibody (Santa Cruz, catalogue #sc-13118, at 1:1000 dilution), rabbit monoclonal GFP antibody (Cell Signaling, catalogue #2956S, at 1:1000 dilution) and rabbit monoclonal NOTCH2 and ITCH antibodies (Cell Signaling, catalogue #4530S, 12117S, both at 1:2000 dilution).

Techniques: Transfection, Western Blot, Knock-Out, Immunoprecipitation, Control, Plasmid Preparation, Incubation, Knockdown

Journal: Nature Communications

Article Title: Plasma membrane-derived extracellular microvesicles mediate non-canonical intercellular NOTCH signaling

doi: 10.1038/s41467-017-00767-2

Figure Lengend Snippet: ARMMs deliver functional NOTCH2 to recipient cells. a Schematic drawing of the transwell assay in which cultured recipient and donor cells were separated by a 0.4 μm membrane. b Western blotting of NOTCH2 (GFP-tagged) in donor and recipient cells. HEK293T cells were transfected with GFP, NOTCH2 delECD -GFP (NOTCH2 used here does not contain the extracellular domain) and ARRDC1-GFP. Twenty-four hours later, the transfected cells were seeded as donor cells in transwells and co-cultured with recipient HEK293T cells. Recipient cells were washed with PBS and lysed for anti-GFP western blotting analysis. GAPDH western blotting was done to ensure equal loading. Asterisk indicates a non-specific protein band. c Expression of NOTCH target genes in recipient cells. Donor HEK293T cells were transfected with GFP, NOTCH2 delECD -GFP or NOTCH2 delECD -GFP plus HA-ARRDC1, and co-cultured with recipient HEK293T cells in transwells for 48 h. RNAs were then extracted from the recipient cells and used for qRT-PCR to measure the expression of HES1 and HES5 genes. d qRT-PCR data showing HES1/HES5 mRNA levels in recipient cells that received ARMMs from control or ARRDC1-KO HEK293T cells. Data represent an average of three independent repeats for each condition. Error bars indicate standard deviation (s.d.). * p < 0.05

Article Snippet: Primary antibodies used in the study include rabbit polyclonal ARRDC1 antibody as described previously , mouse monoclonal CD9 antibody (Santa Cruz, catalogue #sc-13118, at 1:1000 dilution), rabbit monoclonal GFP antibody (Cell Signaling, catalogue #2956S, at 1:1000 dilution) and rabbit monoclonal NOTCH2 and ITCH antibodies (Cell Signaling, catalogue #4530S, 12117S, both at 1:2000 dilution).

Techniques: Functional Assay, Transwell Assay, Cell Culture, Membrane, Western Blot, Transfection, Expressing, Quantitative RT-PCR, Control, Standard Deviation

Journal: Nature Communications

Article Title: Plasma membrane-derived extracellular microvesicles mediate non-canonical intercellular NOTCH signaling

doi: 10.1038/s41467-017-00767-2

Figure Lengend Snippet: Roles of ADAM10 and γ-secretase in NOTCH2 release and activation. a Schematic drawing of NOTCH2 receptor with indicated cleavage sites for ADAM10 and γ-secretase. b Effect of ADAM10 knockdown on NOTCH2 release into ARMMs. Left panel : qRT-PCR data showing siRNA-mediated knockdown of ADAM10 in HEK293T cells. Right panel : Western blot analysis. HEK293T cells were transfected with scrambled control or ADMA10 siRNAs. EVs and cell lysates were collected and subjected to western blotting. c Effect of γ-secretase inhibitor on NOTCH activation in recipient cells. HEK293T donor cells were transfected with NOTCH2 delECD -GFP alone or together with HA-ARRDC1. Donor cells were then co-cultured with recipient HEK293T cells in the absence or presence of 100 nM γ-secretase inhibitor DAPT. qRT-PCR data showed the expression of HES genes in recipient cells. Data represent an average of three independent repeats for each condition. Error bars indicate standard deviation (s.d.). * p < 0.05. d Proposed model for ARMMs-mediated non-canonical NOTCH2 signaling

Article Snippet: Primary antibodies used in the study include rabbit polyclonal ARRDC1 antibody as described previously , mouse monoclonal CD9 antibody (Santa Cruz, catalogue #sc-13118, at 1:1000 dilution), rabbit monoclonal GFP antibody (Cell Signaling, catalogue #2956S, at 1:1000 dilution) and rabbit monoclonal NOTCH2 and ITCH antibodies (Cell Signaling, catalogue #4530S, 12117S, both at 1:2000 dilution).

Techniques: Activation Assay, Knockdown, Quantitative RT-PCR, Western Blot, Transfection, Control, Cell Culture, Expressing, Standard Deviation

Journal: International Journal of Molecular Sciences

Article Title: The Interaction of the Senescent and Adjacent Breast Cancer Cells Promotes the Metastasis of Heterogeneous Breast Cancer Cells through Notch Signaling

doi: 10.3390/ijms22020849

Figure Lengend Snippet: Activation of Notch signaling in both senescent and adjacent non-senescent breast cancer MCF-7 cells. ( A ) Scheme showing the experimental setup. Flow cytometry sorting of collected breast cancer cells after co-culture or monoculture. ( B ) Immunoblot analysis of protein expression of active Notch1 intracellular domain (N1ICD) and the canonical Notch target, HES1, assessed in MCF7-mRFP cells subjected to the indicated treatments. ( C ) RT-qPCR analysis of expression of target genes HES1 and HEY1 and JAG1 in MCF7-mRFP cells subjected to the indicated treatments (error bars indicate mean SD, n = 3 experimental replicates,* p < 0.05, ** p < 0.01). ( D ) Immunoblot analysis of protein expression of active Notch1 intracellular domain (N1ICD) and the canonical Notch1target, HES1, assessed in MCF-7 cells subjected to the indicated treatments. ( E ) RT-qPCR analysis of gene expression of target genes HES1 and HEY1 and the JAG1 ligand of Notch in MCF-7 cells subjected to the indicated treatments (error bars indicate the mean SD, n = 3 experimental replicates,* p < 0.05, ** p < 0.01).

Article Snippet: Antibodies were procured against E-cadherin (BD Biosciences, Lexington, KY, USA), Vimentin (BD Biosciences), β-actin (Sigma-Aldrich, A5228), Notch1 (Cell Signaling Technology, Boston, MA, USA), cleaved Notch1 (Cell Signaling Technology), cyclinA2 (Abcam, Cambridge, UK), Ki67 (GeneTex, GTX16667), Hes1 (Cell Signaling Technology), p21 (Proteintech, Rosemont, IL, USA), β-catenin (BD Biosciences), cyclinD1 (Santa Cruz Biotechnology, CA, USA), and c-Myc(Santa Cruz Biotechnology).

Techniques: Activation Assay, Flow Cytometry, Co-Culture Assay, Western Blot, Expressing, Quantitative RT-PCR, Gene Expression

Journal: International Journal of Molecular Sciences

Article Title: The Interaction of the Senescent and Adjacent Breast Cancer Cells Promotes the Metastasis of Heterogeneous Breast Cancer Cells through Notch Signaling

doi: 10.3390/ijms22020849

Figure Lengend Snippet: N -[(3,5-Difluorophenyl)acetyl]-l-alanyl-2-phenyl]glycine-1,1-dimethylethyl ester (DAPT) inhibits EMT and metastasis of the senescent and adjacent non-senescent breast cancer cells. ( A ) Scheme showing the experimental setup. Flow cytometry analysis of collected breast cancer cells in co-cultures or monocultures treated with or without DAPT (10 μM). ( B ) Immunoblot analysis of protein expression of active Notch1 intracellular domain (N1ICD), E-cadherin, and Vimentin in MCF-7-mRFP and MCF-7 cells treated with or without DAPT. ( C , D ) Wound-healing assay of MCF-7 cells (MCF-7-mRFP and SEN-MCF-7) treated with or without DAPT. ( C ) Representative micrographs of the wound-healing assay are shown. ( D ) Data represent the relative migration ratio of cells per field (error bars indicate mean SD, n = 3 experimental replicates, * p < 0.05). ( E ) Migration assay of MCF-7 cells (MCF-7-mRFP and SEN-MCF-7) treated with or without DAPT. Representative micrographs of migrated cells are shown. Data represent the number of cells derived from mean cell counts of five fields (error bars indicate mean SD, n = 3 experimental replicates, *** p < 0.001). ( F ) Invasion assay of MCF-7 cells (MCF-7-mRFP and SEN-MCF-7) with indicated treatments. Representative micrographs of the migrated cells are shown. Data represent the number of cells derived from mean cell counts of five fields (error bars indicate mean SD, n = 3 experimental replicates, * p < 0.05, ** p < 0.01, *** p < 0.001). ( G ) Immunoblot analysis of protein expression of E-cadherin, Vimentin, N1ICD, and cyclinA2 in T47D, SEN-T47D and Co-culture-T47D (which contains T47D and SEN-T47D). ( H , I ) Wound-healing assay of T47D subjected to the indicated treatments. ( H ) Representative micrographs of the wound-healing assay are shown. ( I ) Data represent the relative migration ratio of cells per field (error bars indicate mean SD, n = 3 experimental replicates, * p < 0.05, ** p < 0.01, *** p < 0.001). ( J ) Migration assays of T47D cells with the indicated treatments. Representative micrographs of migrated cells are shown. Data represent the number of cells derived from mean cell counts of five fields (Error bars indicate mean SD, n = 3 experimental replicates, ** p < 0.01, *** p < 0.001). ( K ) Invasion assay of T47D cells with the indicated treatments. Representative micrographs of migrated cells are shown. Data represent the number of cells derived from mean cell counts of five fields (error bars indicate mean SD, n = 3 experimental replicates, *** p < 0.001). ( L ) Lung metastatic nodules were confirmed by hematoxylin and eosin staining. Scale bars, 250μm. ( M ) The number of visible surface metastatic lesions in lungs was counted (error bars indicate mean SD, n = 6 mice for each group, * p < 0.05, *** p < 0.001).

Article Snippet: Antibodies were procured against E-cadherin (BD Biosciences, Lexington, KY, USA), Vimentin (BD Biosciences), β-actin (Sigma-Aldrich, A5228), Notch1 (Cell Signaling Technology, Boston, MA, USA), cleaved Notch1 (Cell Signaling Technology), cyclinA2 (Abcam, Cambridge, UK), Ki67 (GeneTex, GTX16667), Hes1 (Cell Signaling Technology), p21 (Proteintech, Rosemont, IL, USA), β-catenin (BD Biosciences), cyclinD1 (Santa Cruz Biotechnology, CA, USA), and c-Myc(Santa Cruz Biotechnology).

Techniques: Flow Cytometry, Western Blot, Expressing, Wound Healing Assay, Migration, Derivative Assay, Invasion Assay, Co-Culture Assay, Staining

Journal: Scientific reports

Article Title: DLK proteins modulate NOTCH signaling to influence a brown or white 3T3-L1 adipocyte fate.

doi: 10.1038/s41598-018-35252-3

Figure Lengend Snippet: Figure 1. The stable over-expression of each one of the Notch genes on 3T3-L1 cells enhances adipogenesis. (A) qRT-PCR analysis of the relative mRNA expression levels of the adipocyte markers aP2 and Pparg in differentiated 3T3-L1 cells. (B) qRT-PCR analysis of the relative Notch and Hes1 mRNA expression levels in differentiated 3T3-L1 cells. Representative Western blots (C) and densitometric analysis (D) of each NOTCH receptor expression in 3T3-L1 adipocytes compared to non-differentiated cells. In the case of the Notch1 gene transfectant intracellular NOTCH1 (NICD1) and complete NOTCH1 protein signals are shown. In the case of the Notch2 gene transfectant, the intracellular NOTCH2 (NICD2) protein signal is shown. For the Notch3 gene transfectant, the complete and the intracellular NOTCH3 (NICD3) protein signals are shown. Finally, for the Notch4 gene transfectant, the complete and the intracellular NOTCH4 (NICD4) protein signals are shown. (E) qRT-PCR analysis of the relative aP2 and Pparg mRNA expression levels in differentiated stable Notch1 gene transfectant (L1-N1D), stable Notch2 gene transfectant (L1-N2D), stable Notch3 gene transfectant (L1-N3D), and stable gene Notch4 transfectant (L1-N4D). Data from qRT-PCR assays were previously normalized to P0 mRNA expression levels. The expression of alpha-tubulin was used as a loading control in all Western blots to normalize expression data. Blot signals from empty vector and over-expressing cells were cropped from original blots and delineated with horizontal white spaces (original blots for each protein signal are shown in Supplementary Figure 4). The fold activation or inhibition was calculated relative to the seven-day differentiated non-transfected or empty-vector-transfected cells, which was set arbitrarily at 1. Data are shown as the mean ± SD of at least three biological assays performed in triplicate. The statistical significance calculated by Student’s t-tests is indicated (*p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001).

Article Snippet: Protein Dilution of primary and secondary antibodies Company NOTCH1 Rabbit anti-NOTCH1 C20R (1:1000) Santa Cruz Biotechnology Active NICD1 Rabbit anti-Cleaved NOTCH1 (Val 1744) (1:000) Cell signaling NOTCH2 Goat anti-NOTCH2 M20 (1:500) Santa Cruz Biotechnology NOTCH3 Rabbit anti-NOTCH3 (1:1000) Abcam NOTCH4 Rabbit anti-NOTCH4 (1:1000) Upstate Millipore HA Mouse anti-HA 16B12 (1:5000) Covance DLK1 Rabbit anti-DLK1 (1:1000) Nueda et al.49 DLK2 Rabbit anti-DLK2 (1:500) Proteintech α-tubulin Mouse anti-alpha-Tubulin (1:5000) Sigma Table 2.

Techniques: Over Expression, Quantitative RT-PCR, Expressing, Western Blot, Transfection, Control, Plasmid Preparation, Activation Assay, Inhibition

Journal: Scientific reports

Article Title: DLK proteins modulate NOTCH signaling to influence a brown or white 3T3-L1 adipocyte fate.

doi: 10.1038/s41598-018-35252-3

Figure Lengend Snippet: Figure 2. Effects of stable over-expression of each one of the Notch genes in 3T3-L1 adipocyte browning. (A) Representative microscopy images (400X magnification) of 3T3-L1 adipocytes (L1D) seven days after standard adipogenic induction (48 hours with IBMX and dexamethasone, and 5 days with insulin, see Methods) and non-treated 3T3-L1 cells (L1C). Scale bar (250 μm) is shown. (B) qRT-PCR mRNA expression analysis of the brown adipocyte markers Ucp1, Pgc1a, Gyk, Prdm16, Cidea and Sirt1 in seven-day-differentiated 3T3-L1 cells. qRT-PCR analysis of the relative mRNA expression levels of Ucp1, Pgc1a, Gyk, Prdm16, Cidea and Sirt1 markers in seven-day differentiated Notch1 gene transfectant (L1-N1D) (C), Notch2 gene transfectant (L1-N2D) (D), Notch3 gene transfectant (L1-N3D) (E), Notch4 gene transfectant (L1-N4D) (F). Data from qRT-PCR assays were previously normalized to P0 mRNA expression levels. qPCR analysis of mitochondrial CytB DNA amplification (related to genomic ApoB DNA amplification, see Methods) in seven-day differentiated 3T3-L1 cells over-expressing Notch1 gene (G) and Notch2, Notch3 or Notch4 genes (H). The fold activation or inhibition was calculated relative to the seven-day differentiated non-transfected or empty-vector-transfected cells, which was set arbitrarily at 1. Data are shown as the mean ± SD of at least three biological assays performed in triplicate. The statistical significance of the Student’s t-tests performed is indicated (*p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001).

Article Snippet: Protein Dilution of primary and secondary antibodies Company NOTCH1 Rabbit anti-NOTCH1 C20R (1:1000) Santa Cruz Biotechnology Active NICD1 Rabbit anti-Cleaved NOTCH1 (Val 1744) (1:000) Cell signaling NOTCH2 Goat anti-NOTCH2 M20 (1:500) Santa Cruz Biotechnology NOTCH3 Rabbit anti-NOTCH3 (1:1000) Abcam NOTCH4 Rabbit anti-NOTCH4 (1:1000) Upstate Millipore HA Mouse anti-HA 16B12 (1:5000) Covance DLK1 Rabbit anti-DLK1 (1:1000) Nueda et al.49 DLK2 Rabbit anti-DLK2 (1:500) Proteintech α-tubulin Mouse anti-alpha-Tubulin (1:5000) Sigma Table 2.

Techniques: Over Expression, Microscopy, Quantitative RT-PCR, Expressing, Transfection, DNA Amplification, Activation Assay, Inhibition, Plasmid Preparation

Journal: Scientific reports

Article Title: DLK proteins modulate NOTCH signaling to influence a brown or white 3T3-L1 adipocyte fate.

doi: 10.1038/s41598-018-35252-3

Figure Lengend Snippet: Figure 4. Release of glycerol and lactate to the extracellular medium in 3T3-L1 adipocytes over-expressing Dlk or Notch genes. Relative levels of glycerol released to the extracellular medium in response to isoproterenol from Dlk1 or Dlk2 genes (A), and Notch1, 2, 3 or 4 genes (B) over-expressing adipocytes. (C) Representative microscopy images (400X magnification) of non-transfected (L1C) and transfected 3T3-L1 adipocytes (Empty vectors V1, V2, V3 and V4, and their corresponding over-expressing transfectant) under study. The size of their lipid droplets is showed. Scale bar (80 μm) is shown. Relative levels of lactate in the culture supernatant of differentiated non-transfected 3T3-L1 cells (D), Dlk1 or Dlk2 genes over-expressing adipocytes (E), and each of the Notch genes over-expressing adipocytes (F). The fold activation or inhibition was calculated relative to the seven-day differentiated non-transfected or empty-vector-transfected cells, which was set arbitrarily at 1. Data are shown as the mean ± SD of at least three biological assays performed in triplicate. The statistical significance calculated by Student’s t-tests is indicated (*p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001).

Article Snippet: Protein Dilution of primary and secondary antibodies Company NOTCH1 Rabbit anti-NOTCH1 C20R (1:1000) Santa Cruz Biotechnology Active NICD1 Rabbit anti-Cleaved NOTCH1 (Val 1744) (1:000) Cell signaling NOTCH2 Goat anti-NOTCH2 M20 (1:500) Santa Cruz Biotechnology NOTCH3 Rabbit anti-NOTCH3 (1:1000) Abcam NOTCH4 Rabbit anti-NOTCH4 (1:1000) Upstate Millipore HA Mouse anti-HA 16B12 (1:5000) Covance DLK1 Rabbit anti-DLK1 (1:1000) Nueda et al.49 DLK2 Rabbit anti-DLK2 (1:500) Proteintech α-tubulin Mouse anti-alpha-Tubulin (1:5000) Sigma Table 2.

Techniques: Expressing, Microscopy, Transfection, Activation Assay, Inhibition, Plasmid Preparation

Journal: Scientific reports

Article Title: DLK proteins modulate NOTCH signaling to influence a brown or white 3T3-L1 adipocyte fate.

doi: 10.1038/s41598-018-35252-3

Figure Lengend Snippet: Figure 5. Oxygen consumption rate (OCR) in 3T3-L1 adipocytes over-expressing Dlk and Notch genes. Analysis of the relative oxygen consumption rate (OCR) in non-transfected 3T3-L1 cells (A) and 3T3-L1 cells over-expressing Dlk1 or Dlk2 genes (B), and Notch1 (C), Notch2 (D), Notch3 (E) or Notch4 genes (F). The fold activation or inhibition was calculated relative to the time 0 of seven-day differentiated non-transfected or empty-vector-transfected cells, which was set arbitrarily at 1. Data are shown as the mean ± SD of at least three biological assays performed in triplicate. The statistical significance calculated by Student’s t-tests is indicated at 120 minutes (*p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001).

Article Snippet: Protein Dilution of primary and secondary antibodies Company NOTCH1 Rabbit anti-NOTCH1 C20R (1:1000) Santa Cruz Biotechnology Active NICD1 Rabbit anti-Cleaved NOTCH1 (Val 1744) (1:000) Cell signaling NOTCH2 Goat anti-NOTCH2 M20 (1:500) Santa Cruz Biotechnology NOTCH3 Rabbit anti-NOTCH3 (1:1000) Abcam NOTCH4 Rabbit anti-NOTCH4 (1:1000) Upstate Millipore HA Mouse anti-HA 16B12 (1:5000) Covance DLK1 Rabbit anti-DLK1 (1:1000) Nueda et al.49 DLK2 Rabbit anti-DLK2 (1:500) Proteintech α-tubulin Mouse anti-alpha-Tubulin (1:5000) Sigma Table 2.

Techniques: Expressing, Transfection, Activation Assay, Inhibition, Plasmid Preparation

Journal: Scientific reports

Article Title: DLK proteins modulate NOTCH signaling to influence a brown or white 3T3-L1 adipocyte fate.

doi: 10.1038/s41598-018-35252-3

Figure Lengend Snippet: Figure 7. NOTCH activation and signaling in 3T3-L1 cells stably over-expressing each one of the four NOTCH receptors. (A) qRT-PCR analysis of the relative Hes1 and Hey1 mRNA expression levels in the stable Notch1 gene transfectant (L1-N1), the stable Notch2 gene transfectant (L1-N2), the stable Notch3 gene transfectant (L1-N3), and the stable Notch4 gene transfectant (L1-N4). (B) NOTCH transcriptional activity, as measured by gene reporter luciferase assays, in these four Notch genes stable transfectants. (C) qRT-PCR analysis of the relative individual Notch mRNA expression levels in stable Notch1 gene transfectant (L1-N1), the stable Notch2 gene transfectant (L1-N2), the stable Notch3 gene transfectant (L1-N3), and the stable Notch4 gene transfectant (L1-N4). The relative luciferase activities were always normalized with renilla values and referred to those of control cells. Data in all qRT-PCR assays were normalized to P0 mRNA expression levels. The fold activation or inhibition in all assays is measured relative to the empty vector control, set arbitrarily at 1. Data are shown as the mean ± SD of at least three biological assays performed in triplicate. The statistical significance of Student’s t-tests results is indicated (*p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001).

Article Snippet: Protein Dilution of primary and secondary antibodies Company NOTCH1 Rabbit anti-NOTCH1 C20R (1:1000) Santa Cruz Biotechnology Active NICD1 Rabbit anti-Cleaved NOTCH1 (Val 1744) (1:000) Cell signaling NOTCH2 Goat anti-NOTCH2 M20 (1:500) Santa Cruz Biotechnology NOTCH3 Rabbit anti-NOTCH3 (1:1000) Abcam NOTCH4 Rabbit anti-NOTCH4 (1:1000) Upstate Millipore HA Mouse anti-HA 16B12 (1:5000) Covance DLK1 Rabbit anti-DLK1 (1:1000) Nueda et al.49 DLK2 Rabbit anti-DLK2 (1:500) Proteintech α-tubulin Mouse anti-alpha-Tubulin (1:5000) Sigma Table 2.

Techniques: Activation Assay, Stable Transfection, Expressing, Quantitative RT-PCR, Transfection, Activity Assay, Luciferase, Control, Inhibition, Plasmid Preparation

Journal: Scientific reports

Article Title: DLK proteins modulate NOTCH signaling to influence a brown or white 3T3-L1 adipocyte fate.

doi: 10.1038/s41598-018-35252-3

Figure Lengend Snippet: Figure 8. Feedback modulation among Notch and Dlk gene expression in 3T3-L1 preadipocytes. (A) qRT- PCR analysis of the relative individual Dlk (B) mRNA expression levels in the stable Notch1 gene transfectant (L1-N1), the stable Notch2 gene transfectant (L1-N2), the stable Notch3 gene transfectant (L1-N3), and the stable Notch4 gene transfectant (L1-N4). (B) qRT-PCR analysis of the relative Hes1 and Dlk mRNA expression levels in the stable Hes1 gene transfectant (L1-H1). (C) qRT-PCR analysis of the relative Hes1 and Hey1 mRNA expression levels in the stable Dlk1 gene transfectant (L1-DLK1) and the stable Dlk2 gene transfectant (L1- DLK2). qRT-PCR analysis of the relative Notch (D) and Dlk (E) mRNA expression levels in the stable Dlk1 gene transfectant (L1-DLK1), and the stable Dlk2 gene transfectant (L1-DLK2). In all qRT-PCR assays, data were normalized to P0 mRNA expression levels. The fold activation or inhibition in all assays was measured relative to the empty vector, set arbitrarily at 1. Data are shown as the mean ± SD of at least three biological assays performed in triplicate. The statistical significance of Student’s t-tests results is indicated (*p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001).

Article Snippet: Protein Dilution of primary and secondary antibodies Company NOTCH1 Rabbit anti-NOTCH1 C20R (1:1000) Santa Cruz Biotechnology Active NICD1 Rabbit anti-Cleaved NOTCH1 (Val 1744) (1:000) Cell signaling NOTCH2 Goat anti-NOTCH2 M20 (1:500) Santa Cruz Biotechnology NOTCH3 Rabbit anti-NOTCH3 (1:1000) Abcam NOTCH4 Rabbit anti-NOTCH4 (1:1000) Upstate Millipore HA Mouse anti-HA 16B12 (1:5000) Covance DLK1 Rabbit anti-DLK1 (1:1000) Nueda et al.49 DLK2 Rabbit anti-DLK2 (1:500) Proteintech α-tubulin Mouse anti-alpha-Tubulin (1:5000) Sigma Table 2.

Techniques: Gene Expression, Quantitative RT-PCR, Expressing, Transfection, Activation Assay, Inhibition, Plasmid Preparation