Journal: The Journal of Cell Biology

Article Title: Basement membrane assembly of the integrin α8β1 ligand nephronectin requires Fraser syndrome–associated proteins

doi: 10.1083/jcb.201203065

Figure Lengend Snippet: In situ binding of recombinant integrins to frozen sections of mouse embryonic tissues. (A–C) Recombinant integrin α8β1 (green) bound to E15.5 dorsal skin (A), lung (B), and choroid plexus (C) cryosections. The top and bottom panels show fluorescence images for wild-type and Qbrick −/− littermates, respectively. (D–F) Integrins α3β1 (D), α6β4 (E), and α7X2β1 (F) bound to dorsal skin cryosections from wild-type (top) and Qbrick −/− (bottom) littermates were visualized (green). (G and H) Integrin α8β1 (green) bound to dorsal skin cryosections from Frem2 my /+ and Frem2 my / my (G) and Grip1 eb /+ and Grip1 eb / eb (H) mice. The top and bottom panels show fluorescence images for control heterozygotes and homozygotes, respectively. The BMs were counterstained with an anti–laminin-γ1 chain antibody (magenta). Immunoreactivity against QBRICK (green) is also shown. Arrowheads indicate the location of BMs. The asterisks indicate nonspecific binding of recombinant integrins or the anti-QBRICK antibody to the cornified epithelium. Bars, 20 µm.

Article Snippet: The primary antibodies used were: rabbit anti-laminin (L9393; Sigma-Aldrich), goat anti–mouse integrin α8 (R&D Systems), rabbit anti-nephronectin , and rabbit anti-MAEG ( ).

Techniques: In Situ, Binding Assay, Recombinant, Fluorescence, Control

Journal: The Journal of Cell Biology

Article Title: Basement membrane assembly of the integrin α8β1 ligand nephronectin requires Fraser syndrome–associated proteins

doi: 10.1083/jcb.201203065

Figure Lengend Snippet: Impaired binding of integrin α8β1 in the developing kidney of Qbrick −/− mice. (A) Unilateral renal agenesis observed in Qbrick −/− mice. Bar, 5 mm. (B and C) The left panels show integrin α8β1 (green) bound to the BMs of the E10.5 mesonephric duct (B) and E11.5 ureteric bud (C) of wild-type (top) and Qbrick −/− (bottom) embryos. The right panels show the BMs counterstained with an anti–laminin-γ1 chain antibody (magenta). (D and E) QBRICK immunofluorescence (green) in the developing kidney of wild-type (top) and Qbrick −/− (bottom) embryos at E10.5 (D) and E11.5 (E). The BMs were counterstained with an anti–laminin-γ1 chain antibody (magenta). The open and closed arrowheads indicate the mesonephric duct and ureteric bud, respectively. The asterisks indicate nonspecific binding of the anti-QBRICK antibody to the mesenchyme. Bars, 50 µm. (F) The expression levels of Gdnf and Pax2 transcripts in the E11.5 metanephros of Qbrick +/− control (open bars) and Qbrick −/− (shaded bars) littermates were determined by quantitative RT-PCR and normalized by the expression level of Gapdh . The expression level in control mice was set at 1. Each bar represents the mean ± SD (error bars; n = 3). **, P < 0.01, significant difference by Student’s t test.

Article Snippet: The primary antibodies used were: rabbit anti-laminin (L9393; Sigma-Aldrich), goat anti–mouse integrin α8 (R&D Systems), rabbit anti-nephronectin , and rabbit anti-MAEG ( ).

Techniques: Binding Assay, Immunofluorescence, Expressing, Control, Quantitative RT-PCR

Journal: The Journal of Cell Biology

Article Title: Basement membrane assembly of the integrin α8β1 ligand nephronectin requires Fraser syndrome–associated proteins

doi: 10.1083/jcb.201203065

Figure Lengend Snippet: Generation of Qbrick RGE/RGE mice. (A) Schematic representation of the targeted mutation of Qbrick . Open boxes represent exons. The knock-in construct was designed to replace exon 4, which contains the codon encoding Asp207, with a mutated exon (open box with asterisk) in which the codon encoding Asp207 was substituted with a codon encoding Glu207. The probes used for Southern blotting are indicated by bold lines. N, NcoI restriction site; TK, thymidine kinase. (B) Direct DNA sequencing of the wild-type and Qbrick RGE/RGE genomes. The numbers indicate the positions of the amino acids in the primary sequence of QBRICK protein. (C) Southern blot analyses of genomic DNA from wild-type, Qbrick RGEneo/+ , and Qbrick RGE/+ offspring after digestion with NcoI. Fragments of 5.3 and 7.7 kbp are detected with probe 1 and probe 2, respectively, indicating that the expected homologous recombination occurred. The detection of a 6.1-kbp fragment with probe 2 indicates that the neomycin resistance gene has been removed from the Qbrick RGEneo allele by the Cre-loxP system. (D and E) Immunofluorescence staining (green) for QBRICK in E15.5 dorsal skin (D) and E11.5 ureteric buds (E). The BMs were counterstained with an anti–laminin-γ1 chain antibody (magenta). (F–H) Qbrick RGE/RGE mice develop normally without any defects in the kidneys (asterisks in F), eyelids (G), and digits (H). (I and J) Binding of recombinant integrin α8β1 (green) to E15.5 dorsal skin (I) and E11.5 ureteric buds (J). The BMs were counterstained with an anti–laminin-γ1 chain antibody (magenta). In panels D, E, I, and J, the top and bottom panels show fluorescence images for wild-type and Qbrick RGE/RGE littermates, respectively. The expression levels of QBRICK are comparable between wild-type and Qbrick RGE/RGE littermates. It should be noted that the binding of integrin α8β1 to the epidermal BMs of the E15.5 dorsal skin (I) as well as the E11.5 ureteric buds (J) is comparable between wild-type and Qbrick RGE/RGE littermates. Bars: (D and I) 20 µm; (E and J) 50 µm; (F) 5 mm.

Article Snippet: The primary antibodies used were: rabbit anti-laminin (L9393; Sigma-Aldrich), goat anti–mouse integrin α8 (R&D Systems), rabbit anti-nephronectin , and rabbit anti-MAEG ( ).

Techniques: Mutagenesis, Knock-In, Construct, Southern Blot, DNA Sequencing, Sequencing, Homologous Recombination, Immunofluorescence, Staining, Binding Assay, Recombinant, Fluorescence, Expressing

Journal: The Journal of Cell Biology

Article Title: Basement membrane assembly of the integrin α8β1 ligand nephronectin requires Fraser syndrome–associated proteins

doi: 10.1083/jcb.201203065

Figure Lengend Snippet: Impaired expression of nephronectin and MAEG in FS model mice. (A–F) Immunofluorescence staining (green) for nephronectin (A, C, and E) and MAEG (B, D, and F) in the dorsal skin of Qbrick −/− (A and B), Frem2 my / my (C and D), and Grip1 eb / eb (E and F) mice, and their control wild-type or heterozygous littermates. (G–J) Immunofluorescence staining (green) for nephronectin (G and H) and MAEG (I and J) at the E10.5 mesonephric duct (G and I; open arrowheads) and E11.5 ureteric bud (H and J; closed arrowheads) in wild-type (top) and Qbrick −/− (bottom) littermates. The BMs were counterstained with an anti–laminin-γ1 chain antibody (magenta). Bars: (A–F) 20 µm; (G–J) 50 µm. (K and L) Immunoblot detection of laminin β1 and γ1 chains (LN), integrin α8 (Intα8), nephronectin (Npnt), and MAEG in protein extracts from the kidney (K) and skin (L) of E15.5 Qbrick −/− mice and their wild-type littermates. (M–O) Immunoblot signal intensities of laminin, integrin α8, nephronectin, and MAEG in the kidney (left) and skin (right) of E15.5 wild-type (open bars) and Qbrick −/− (shaded bars; M), E15.5 Frem2 my /+ (open bars) and Frem2 my / my (shaded bars; N), and E17.5 Grip1 eb /+ (open bars) and Grip1 eb / eb (shaded bars; O) mice. The signal levels in control mice were set at 1. Each bar represents the mean ± SD (error bars; n = 3–6). *, P < 0.05; **, P < 0.01; ***, P < 0.001, significant differences by Student’s t tests. (P) Titration curves of recombinant integrin α8β1 bound to the GST-fused NV domain of QBRICK (open triangles), GST-fused RGD linker segment of nephronectin (open diamonds), GST-fused RGD linker segment of MAEG (open squares), and GST (closed circles). Each point represents the mean ± SEM ( n = 3).

Article Snippet: The primary antibodies used were: rabbit anti-laminin (L9393; Sigma-Aldrich), goat anti–mouse integrin α8 (R&D Systems), rabbit anti-nephronectin , and rabbit anti-MAEG ( ).

Techniques: Expressing, Immunofluorescence, Staining, Control, Western Blot, Titration, Recombinant

Journal: The Journal of Cell Biology

Article Title: Basement membrane assembly of the integrin α8β1 ligand nephronectin requires Fraser syndrome–associated proteins

doi: 10.1083/jcb.201203065

Figure Lengend Snippet: The binding of integrin α8β1 to BMs is not compromised in Maeg ΔRGD mice. (A) Schematic views of MAEG and MAEG ΔRGD , a deletion mutant lacking the linker segment. The sites recognized by the anti-MAEG antibodies (Anti-linker and Anti-MAM) are also shown. (B) Schematic representation of the targeted mutation of Maeg . Open boxes represent exons. The targeting construct was designed to replace exons 9 and 10, which encode the linker region, with PGKneo. The probes used for Southern blotting are indicated by bold lines. B, BamHI restriction site; TK, thymidine kinase. (C) Southern blot analysis of genomic DNA from wild-type, Maeg ΔRGD/+ , and Maeg ΔRGD/Y mice digested with BamHI. The detection of a 6.3-kbp fragment instead of an 8.2-kbp fragment indicates the presence of the mutated allele. (D) Direct DNA sequencing of the Maeg ΔRGD/Y -derived RT-PCR product demonstrating that the open reading frame has not shifted. The product encodes a polypeptide lacking amino acids 258–391 of the 550–amino-acid full-length sequence. The boundary between exons 8 and 11 is shown by the red broken line. The numbers indicate the positions of the corresponding amino acids in the wild-type MAEG sequence. (E and F) In E15.5 dorsal skin epidermal BMs, MAEG is comparably detected by immunofluorescence (green) in both Maeg +/Y and Maeg ΔRGD/Y littermates with the anti-MAM antibody (E), but is only detected in Maeg +/Y embryos by the anti-linker antibody (F). (G, H, and K) Immunofluorescence staining for QBRICK (G, green) and nephronectin (H, green), and binding of recombinant integrin α8β1 (K, green) to epidermal BMs of the E15.5 dorsal skin are comparable between Maeg +/Y and Maeg ΔRGD/Y littermates. The BMs were counterstained with an anti–laminin-γ1 chain antibody (magenta). In each set of panels, the top and bottom panels show fluorescence images for wild-type Maeg +/Y and Maeg ΔRGD/Y littermates, respectively. Bars, 20 µm. (I and J) Immunoblot signal intensities of laminin β1 and γ1 chains (LN), integrin α8 (Intα8), and nephronectin (Npnt) in the kidney (I) and skin (J) of E15.5 control Maeg +/Y (open bars) and Maeg ΔRGD/Y (shaded bars) mice. The signal levels in control mice were set at 1. Each bar represents the mean ± SD (error bars; n = 3).

Article Snippet: The primary antibodies used were: rabbit anti-laminin (L9393; Sigma-Aldrich), goat anti–mouse integrin α8 (R&D Systems), rabbit anti-nephronectin , and rabbit anti-MAEG ( ).

Techniques: Binding Assay, Mutagenesis, Construct, Southern Blot, DNA Sequencing, Derivative Assay, Reverse Transcription Polymerase Chain Reaction, Sequencing, Immunofluorescence, Staining, Recombinant, Fluorescence, Western Blot, Control

Journal: The Journal of Cell Biology

Article Title: Basement membrane assembly of the integrin α8β1 ligand nephronectin requires Fraser syndrome–associated proteins

doi: 10.1083/jcb.201203065

Figure Lengend Snippet: A model for renal defects in FS. In the developing metanephros, QBRICK and other FS-associated proteins associate with nephronectin in the sublamina densa region and secure its localization at BMs. Metanephric mesenchymal cells interact with nephronectin through integrin α8β1. Stimulation of integrin α8β1 induces Gdnf , which is necessary for further development of the metanephros (left). In FS, nephronectin is not stably deposited at BMs, owing to the loss of FS-associated proteins, and is eventually degraded. The loss of nephronectin leads to failure of Gdnf induction and subsequent metanephric development (right).

Article Snippet: The primary antibodies used were: rabbit anti-laminin (L9393; Sigma-Aldrich), goat anti–mouse integrin α8 (R&D Systems), rabbit anti-nephronectin , and rabbit anti-MAEG ( ).

Techniques: Stable Transfection

Journal: The Journal of Veterinary Medical Science

Article Title: Tensin2-deficient mice on FVB/N background develop severe glomerular disease

doi: 10.1292/jvms.15-0442

Figure Lengend Snippet: Dual immunofluorescence study for a mesangial marker, integrin α8 and laminin α2 in 3-week-old female FVB and FVBGN mice. Mesangial process invasion of the glomerular capillary tufts was observed in FVBGN mice. Laminin α2 localized to the mesangium and the GBM in FVBGN mice. Arrows denote area of dual immunostaining in the glomerular capillary loop. Scale bars=50 µ m.

Article Snippet: The primary antibodies used were as follows: anti-laminin α1 rat monoclonal and anti-fibronectin rabbit polyclonal antibodies at 1:100 (Millipore, Billerica, MA, U.S.A.), anti-laminin α2, β1 and γ1 rat monoclonal and anti-CD151 rabbit polyclonal antibodies at 1:200 (Santa Cruz), anti-laminin α5 and β2 rabbit polyclonal antibodies (Sigma, St. Louis, MO, U.S.A.), anti-α1, α2, α3, α4 and α5 chains of collagen IV rat monoclonal antibodies at 1:100 (Chondrex, Redmond, WA, U.S.A.), anti-phospho-FAK rabbit polyclonal antibody at 1:100 (Abcam, Cambridge, U.K.) and anti-integrin α8 goat polyclonal antibody at 1:200 (R&D Systems, Minneapolis, MN, U.S.A.).

Techniques: Immunofluorescence, Marker, Immunostaining

Journal: PLoS ONE

Article Title: Laminin α2-Mediated Focal Adhesion Kinase Activation Triggers Alport Glomerular Pathogenesis

doi: 10.1371/journal.pone.0099083

Figure Lengend Snippet: The same mice as in Figure 8 were dual immunostained with the GBM marker, laminin α5, and the mesangial cell marker, integrin α8. Arrowheads in panel C denote regions where invasion of the capillary loops by mesangial processes is evident (panels B and C, inserts). This characteristic is markedly reduced in the TAE226-treated glomeruli where integrin α8 immunostaining is restricted to the mesangial angles (panels E and F. inserts). Transmission electron microscopic analysis shows that TAE226 treatment (Panel I) reduces the ultrastructural damage to the GBM normally present by 7 weeks of age in this model (panel H). Panel J is provided to indicate that amelioration of GBM dysmorphology is generally observed. Panel K shows that treatment of primary cultured mesangial cells with TAE226 significantly reduces their migratory potential relative to untreated cells. Panel L shows a dose response for FAK inhibition by TAE226 in cultured mesangial cells. Panel F, scale bar = 15 µm; Panel J, scale bar = 2 µm.

Article Snippet: The dual stain consisting of rat anti-mouse Laminin-α2 antibody (Sigma-Aldrich, St. Louis, MO) at 1∶200 and rabbit anti-mouse phospho-FAK 397 antibody at 1∶25 as well as the dual stain of goat anti-mouse Integrin α8 antibody (R & D Systems, Minneapolis, MN) at 1∶1000 and rabbit anti-mouse Laminin-5 antibody, at 1∶1000 were diluted in 0.3% PBST+5% FBS.

Techniques: Marker, Immunostaining, Transmission Assay, Cell Culture, Inhibition