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: