Journal: Frontiers in Physiology

Article Title: Aestivation Induces Changes in the mRNA Expression Levels and Protein Abundance of Two Isoforms of Urea Transporters in the Gills of the African Lungfish, Protopterus annectens

doi: 10.3389/fphys.2017.00071

Figure Lengend Snippet: Primers used for PCR, RACE, and qPCR of urea transporter a2a ( ut-a2a ) and urea transporter a2b ( ut-a2b ) from the gills of Protopterus annectens .

Article Snippet: The anti-Ut-a2a and anti-Ut-a2b (16.7 μg each) antibodies were pre-incubated with their corresponding immunizing peptide (83.5 μg each; Genscript) at 25°C for 1 h and subsequently used for the peptide competition assay.

Techniques: Sequencing

Journal: Frontiers in Physiology

Article Title: Aestivation Induces Changes in the mRNA Expression Levels and Protein Abundance of Two Isoforms of Urea Transporters in the Gills of the African Lungfish, Protopterus annectens

doi: 10.3389/fphys.2017.00071

Figure Lengend Snippet: A multiple amino acid alignment of the isoforms of urea transporter (Ut-a2a and Ut-a2b) from Protopterus annectens with Latimeria chalumnae Ut-a2 (XP_006007026.1), Squalus acanthias Ut-a2 (AAF66072.1) , Opsanus beta Ut-a2 (AAD53268.2) , Rhinella marina Ut-a2 (BAE16706.1) , Homo sapiens UT-A2 (CAA65657.1) and UT-B1 (CAB60834.1), and Desulfovibrio vulgaris Ut (Q72CX3) . Identical amino acid residues are indicated by asterisks, strongly similar amino acids are indicated by colons and weakly similar amino acids are indicated by periods. The conserved LPXXTXPF motifs are underlined. The potential urea binding sites are indicated by open triangles. The dotted box denotes the UT-B signature ALE domain. Potential N -glycosylation and phosphorylation sites are indicated by open and shaded arrows, respectively. The predicted transmembrane domains (TM1-10) of Ut-a2a and Ut-a2b of P. annectens are indicated by open boxes and were predicted using MEMSATS and MEMSAT-SVA provided by PSIPRED protein structure prediction server.

Article Snippet: The anti-Ut-a2a and anti-Ut-a2b (16.7 μg each) antibodies were pre-incubated with their corresponding immunizing peptide (83.5 μg each; Genscript) at 25°C for 1 h and subsequently used for the peptide competition assay.

Techniques: Binding Assay, Glycoproteomics, Phospho-proteomics

Journal: Frontiers in Physiology

Article Title: Aestivation Induces Changes in the mRNA Expression Levels and Protein Abundance of Two Isoforms of Urea Transporters in the Gills of the African Lungfish, Protopterus annectens

doi: 10.3389/fphys.2017.00071

Figure Lengend Snippet: A dendrogram of urea transporters (Ut/UT) including Ut-a2a and Ut-a2b of Protopterus annectens . Strongylocentrotus purpuratus Ut is used as outgroup for the dendrogram. Numbers presented at each branch point represent bootstrap values from 100 replicates.

Article Snippet: The anti-Ut-a2a and anti-Ut-a2b (16.7 μg each) antibodies were pre-incubated with their corresponding immunizing peptide (83.5 μg each; Genscript) at 25°C for 1 h and subsequently used for the peptide competition assay.

Techniques:

Journal: Frontiers in Physiology

Article Title: Aestivation Induces Changes in the mRNA Expression Levels and Protein Abundance of Two Isoforms of Urea Transporters in the Gills of the African Lungfish, Protopterus annectens

doi: 10.3389/fphys.2017.00071

Figure Lengend Snippet: Gene expression of (A) urea transporter a2a ( ut-a2a ) and (B) urea transporter a2b ( ut-a2b ) in the eye (E), brain (Br), gills (Gi), heart (H), liver (Li), spleen (Sp), pancreas (P), gut (Gu), kidney (K), Lung (Lu), muscle (M), and skin (Sk) of P. annectens kept in fresh water.

Article Snippet: The anti-Ut-a2a and anti-Ut-a2b (16.7 μg each) antibodies were pre-incubated with their corresponding immunizing peptide (83.5 μg each; Genscript) at 25°C for 1 h and subsequently used for the peptide competition assay.

Techniques: Gene Expression

Journal: Frontiers in Physiology

Article Title: Aestivation Induces Changes in the mRNA Expression Levels and Protein Abundance of Two Isoforms of Urea Transporters in the Gills of the African Lungfish, Protopterus annectens

doi: 10.3389/fphys.2017.00071

Figure Lengend Snippet: Absolute quantification of mRNA (×10 2 copies of transcripts per ng total RNA) of urea transporter a2b ( ut-a2b ) in the gills of Protopterus annectens kept in fresh water on day 0 (FW; control), after 6 days (d; induction phase) or 6 months (mon; maintenance phase) of aestivation, or after 1 or 3 d of arousal (Ar) from 6 mon of aestivation . Results represent means ± S.E.M ( N = 4).

Article Snippet: The anti-Ut-a2a and anti-Ut-a2b (16.7 μg each) antibodies were pre-incubated with their corresponding immunizing peptide (83.5 μg each; Genscript) at 25°C for 1 h and subsequently used for the peptide competition assay.

Techniques: Quantitative Proteomics, Control

Journal: Frontiers in Physiology

Article Title: Aestivation Induces Changes in the mRNA Expression Levels and Protein Abundance of Two Isoforms of Urea Transporters in the Gills of the African Lungfish, Protopterus annectens

doi: 10.3389/fphys.2017.00071

Figure Lengend Snippet: Protein abundance of urea transporter a2b (Ut-a2b) in the gills of Protopterus annectens kept in fresh water on day 0 (FW; control), after 6 days (d; induction phase) or 6 months (mon; maintenance phase) of aestivation, or after 1 or 3 d of arousal (Ar) from 6 mon of aestivation . (A) An example of immunoblot of Ut-a2b (left) and Ut-a2b pre-incubated with immunising peptide for the peptide competition assay (PCA; right). (B) The protein abundance of Ut-a2b expressed as arbitrary densitometric units per 200 μg protein. Results represent mean ± S.E.M . ( N = 3). Means not sharing the same letter are significantly different ( P < 0.05).

Article Snippet: The anti-Ut-a2a and anti-Ut-a2b (16.7 μg each) antibodies were pre-incubated with their corresponding immunizing peptide (83.5 μg each; Genscript) at 25°C for 1 h and subsequently used for the peptide competition assay.

Techniques: Quantitative Proteomics, Control, Western Blot, Incubation, Competitive Binding Assay

Journal: PLoS ONE

Article Title: Lymphangiogenesis and Angiogenesis in Abdominal Aortic Aneurysm

doi: 10.1371/journal.pone.0089830

Figure Lengend Snippet: A , Elastica van Gieson staining of abdominal aortic aneurysm (AAA). Immunohistochemistry for podoplanin ( B ) and macrophages ( C ), CD19 ( E ), and myeloperoxidase (MPO) of the AAA wall. B , Lymphatic microvessels in the intima/media of the AAA (red dotted line encircling lymphatic microvessels). C , Macrophages infiltration around/within lymphatic microvessels in the intima/media or in adventitia (red square: macrophages in intima/media, black square: macrophages in adventitia). D , CD3-positive T cells infiltration around/within lymphatic microvessels in the intima/media or in adventitia. E , CD19-positive B cells infiltration around/within lymphatic microvessels in the intima/media or in adventitia. F , MPO-positive neutrophils infiltration in the intima/media or in adventitia. G–P , Double immunofluorescence staining of macrophages infiltrating the intima/media (In) and adventitia (Ad). G–P , Macrophage: green, CD11b ( G )/LYVE-1 ( H )/VEGF-C ( I )/MMP-9 ( J )/TGF-β1 ( K )/IL-4 ( L )/IL-8 ( M )/MIP-1α ( N )/IFN-γ ( O )/MCP-1 ( P ): red, DAPI: blue. LYVE-1, VEGF-C, MMP-9, TGF-β1, IL-4, IL-8, MIP-1α, and MCP-1 were expressed in the CD11b-positive macrophages in the intima/media, but not by macrophages in adventitia. Q , Double immunofluorescence staining of T-cells in intima/media and inflammatory cytokines. CD3: green, VEGF-C/MMP-9/TGF-β1/IL-4/IL-8/MIP-1α/IFN-γ/MCP-1: red, DAPI: blue. TGF-β1, IL-4, and IFN-γ were expressed in CD3-positive T lymphocytes in the intima/media. R , Double immunofluorescence staining of B lymphocytes in the intima/media and inflammatory cytokines. CD19: green, VEGF-C/MMP-9/TGF-β1/IL-4/IL-8/MIP-1α/IFN-γ/MCP-1: red, DAPI: blue. These inflammatory cytokines were not expressed in CD19-positive B lymphocytes. S , Double immunofluorescence staining of neutrophils in intima/media and inflammatory cytokines. MPO: green, VEGF-C/MMP-9/TGF-β1/IL-4/IL-8/MIP-1α/IFN-γ/MCP-1: red, DAPI: blue. These inflammatory cytokines were not expressed in MPO-positive neutrophils. Scale bars indicated 100 µm ( A–F ) and 10 µm ( G–S ).

Article Snippet: The following primary antibodies were used: mouse monoclonal antibody against podoplanin (1∶200, DakoCytomation, Glostrup, Denmark), rabbit polyclonal antibody against Prox-1 (1∶2000, Millipore, MA, USA), rabbit polyclonal antibody against the N-terminus of human alpha smooth muscle isoform of actin (1∶25, Thermo Scientific Japan, Tokyo, Japan), mouse monoclonal antibody against human HIF-1α (1∶100, Novus Biologicals, CO, USA), rabbit monoclonal antibody against human CD11b (1∶250, Millipore, MA, USA), mouse monoclonal antibody against human macrophages (1∶100, AbD Serotec, Oxford, UK), rabbit polyclonal antibody against human LYVE-1 (1∶100, Relia Tech, Braunschweig, Germany), rabbit polyclonal antibody against human VEGF-C (1∶50, Abcam, Tokyo, Japan), rabbit polyclonal antibody against human matrix metalloproteinase (MMP)-9 (1∶100, Abnova, Taipei, Taiwan), mouse monoclonal antibody against human CD3 (1∶100, LifeSpan Biosciences, Seattle, WA), mouse monoclonal antibody against human CD19 (1∶50, Santa Cruz Biotechnology, CA, USA), mouse monoclonal antibody against human myeloperoxidase (MPO) (1∶50, Santa Cruz Biotechnology, CA, USA), rabbit polyclonal antibody against transforming growth factor beta-1 (TGF-β1) (1∶50, Abbiotec, CA, USA), rabbit polyclonal antibody against human interleukin-4 (IL-4) (1∶50, Biozol, Munich, Germany), rabbit polyclonal antibody against human interleukin-8 (IL-8) (1∶50, Abnova, Taipei, Taiwan), rabbit polyclonal antibody against human macrophage inflammatory protein-1α (MIP-1α) (1∶50, Spring bioscience, CA, USA), rabbit polyclonal antibody against human interferon-γ (IFN-γ) (1∶50, Santa Cruz Biotechnology, CA, USA), and rabbit polyclonal antibody against human monocyte chemotactic protein-1 (MCP-1) (1∶50, Abnova, Taipei, Taiwan).

Techniques: Staining, Immunohistochemistry, Double Immunofluorescence Staining

Journal: PLoS ONE

Article Title: Lymphangiogenesis and Angiogenesis in Abdominal Aortic Aneurysm

doi: 10.1371/journal.pone.0089830

Figure Lengend Snippet: A , Elastica van Gieson staining of abdominal aortic aneurysm (AAA). Immunohistochemistry for podoplanin ( B ) and macrophages ( C ), CD19 ( E ), and myeloperoxidase (MPO) of the AAA wall. B , Lymphatic microvessels in the intima/media of the AAA (red dotted line encircling lymphatic microvessels). C , Macrophages infiltration around/within lymphatic microvessels in the intima/media or in adventitia (red square: macrophages in intima/media, black square: macrophages in adventitia). D , CD3-positive T cells infiltration around/within lymphatic microvessels in the intima/media or in adventitia. E , CD19-positive B cells infiltration around/within lymphatic microvessels in the intima/media or in adventitia. F , MPO-positive neutrophils infiltration in the intima/media or in adventitia. G–P , Double immunofluorescence staining of macrophages infiltrating the intima/media (In) and adventitia (Ad). G–P , Macrophage: green, CD11b ( G )/LYVE-1 ( H )/VEGF-C ( I )/MMP-9 ( J )/TGF-β1 ( K )/IL-4 ( L )/IL-8 ( M )/MIP-1α ( N )/IFN-γ ( O )/MCP-1 ( P ): red, DAPI: blue. LYVE-1, VEGF-C, MMP-9, TGF-β1, IL-4, IL-8, MIP-1α, and MCP-1 were expressed in the CD11b-positive macrophages in the intima/media, but not by macrophages in adventitia. Q , Double immunofluorescence staining of T-cells in intima/media and inflammatory cytokines. CD3: green, VEGF-C/MMP-9/TGF-β1/IL-4/IL-8/MIP-1α/IFN-γ/MCP-1: red, DAPI: blue. TGF-β1, IL-4, and IFN-γ were expressed in CD3-positive T lymphocytes in the intima/media. R , Double immunofluorescence staining of B lymphocytes in the intima/media and inflammatory cytokines. CD19: green, VEGF-C/MMP-9/TGF-β1/IL-4/IL-8/MIP-1α/IFN-γ/MCP-1: red, DAPI: blue. These inflammatory cytokines were not expressed in CD19-positive B lymphocytes. S , Double immunofluorescence staining of neutrophils in intima/media and inflammatory cytokines. MPO: green, VEGF-C/MMP-9/TGF-β1/IL-4/IL-8/MIP-1α/IFN-γ/MCP-1: red, DAPI: blue. These inflammatory cytokines were not expressed in MPO-positive neutrophils. Scale bars indicated 100 µm ( A–F ) and 10 µm ( G–S ).

Article Snippet: The following primary antibodies were used: mouse monoclonal antibody against podoplanin (1∶200, DakoCytomation, Glostrup, Denmark), rabbit polyclonal antibody against Prox-1 (1∶2000, Millipore, MA, USA), rabbit polyclonal antibody against the N-terminus of human alpha smooth muscle isoform of actin (1∶25, Thermo Scientific Japan, Tokyo, Japan), mouse monoclonal antibody against human HIF-1α (1∶100, Novus Biologicals, CO, USA), rabbit monoclonal antibody against human CD11b (1∶250, Millipore, MA, USA), mouse monoclonal antibody against human macrophages (1∶100, AbD Serotec, Oxford, UK), rabbit polyclonal antibody against human LYVE-1 (1∶100, Relia Tech, Braunschweig, Germany), rabbit polyclonal antibody against human VEGF-C (1∶50, Abcam, Tokyo, Japan), rabbit polyclonal antibody against human matrix metalloproteinase (MMP)-9 (1∶100, Abnova, Taipei, Taiwan), mouse monoclonal antibody against human CD3 (1∶100, LifeSpan Biosciences, Seattle, WA), mouse monoclonal antibody against human CD19 (1∶50, Santa Cruz Biotechnology, CA, USA), mouse monoclonal antibody against human myeloperoxidase (MPO) (1∶50, Santa Cruz Biotechnology, CA, USA), rabbit polyclonal antibody against transforming growth factor beta-1 (TGF-β1) (1∶50, Abbiotec, CA, USA), rabbit polyclonal antibody against human interleukin-4 (IL-4) (1∶50, Biozol, Munich, Germany), rabbit polyclonal antibody against human interleukin-8 (IL-8) (1∶50, Abnova, Taipei, Taiwan), rabbit polyclonal antibody against human macrophage inflammatory protein-1α (MIP-1α) (1∶50, Spring bioscience, CA, USA), rabbit polyclonal antibody against human interferon-γ (IFN-γ) (1∶50, Santa Cruz Biotechnology, CA, USA), and rabbit polyclonal antibody against human monocyte chemotactic protein-1 (MCP-1) (1∶50, Abnova, Taipei, Taiwan).

Techniques: Staining, Immunohistochemistry, Double Immunofluorescence Staining