Journal: Critical Care

Article Title: Type XVIII collagen degradation products in acute lung injury

doi: 10.1186/cc7779

Figure Lengend Snippet: Schematic representation of the human collagen XVIII variants, termed as SHORT, MIDDLE and LONG/FZ. Collagenous sequences are shown in white. Non-collagenous (NC) amino terminal sequences common to all variants are shown in black. Non-collagenous amino terminal sequences common to the two long variants are shown in grey. A non-collagenous amino terminal sequence specific to the LONG/FZ variant is marked with waves. The amino acid lengths (aa) and the predicted molecular masses (kDa) of these sequences are given, as are those of the full length type XVIII collagen molecules. Signal sequences are indicated by vertical and horizontal hatching. The epitopes of the antibodies used in this work are shown by black horizontal lines. The molecular masses of some endostatin-containing carboxy-terminal fragments are also indicated.

Article Snippet: A 25 μl sample of BALF was loaded onto polyacrylamide gel and the proteins were separated on a 7 to 12% SDS-PAGE under reducing conditions, electrotransferred to a nitrocellulose membrane (Protran; Schleicher & Schuell, Dassel, Germany) and probed with rabbit polyclonal antibodies against endostatin (HES.6) [ ] and human collagen XVIII (anti-all huXVIII QH4818 and anti-long huXVIII, QH1415.7) [ ], all used at a concentration 1 μg/ml in 5% fat-free milk powder in 1 × PBS, followed by a horseradish peroxidase-conjugated goat anti-rabbit antibody (Bio-Rad, Abingdon, UK).

Techniques: Sequencing, Variant Assay

Journal: Critical Care

Article Title: Type XVIII collagen degradation products in acute lung injury

doi: 10.1186/cc7779

Figure Lengend Snippet: Immunoprecipitation with anti-ALL antibody and detection with anti-LONG huXVIII antibody. Elevated type XVIII collagen precursors in the plasma of ALI patients compared with normal controls is shown. ALI = acute lung injury.

Article Snippet: A 25 μl sample of BALF was loaded onto polyacrylamide gel and the proteins were separated on a 7 to 12% SDS-PAGE under reducing conditions, electrotransferred to a nitrocellulose membrane (Protran; Schleicher & Schuell, Dassel, Germany) and probed with rabbit polyclonal antibodies against endostatin (HES.6) [ ] and human collagen XVIII (anti-all huXVIII QH4818 and anti-long huXVIII, QH1415.7) [ ], all used at a concentration 1 μg/ml in 5% fat-free milk powder in 1 × PBS, followed by a horseradish peroxidase-conjugated goat anti-rabbit antibody (Bio-Rad, Abingdon, UK).

Techniques: Immunoprecipitation, Clinical Proteomics

Journal: Critical Care

Article Title: Type XVIII collagen degradation products in acute lung injury

doi: 10.1186/cc7779

Figure Lengend Snippet: Western blot by anti-ALL huXVIII antibody. It shows elevated type XVIII collagen precursors in the bronchoalveolar lavage fluid (BALF) of patients with acute lung injury compared with normal controls. ALI = acute lung injury.

Article Snippet: A 25 μl sample of BALF was loaded onto polyacrylamide gel and the proteins were separated on a 7 to 12% SDS-PAGE under reducing conditions, electrotransferred to a nitrocellulose membrane (Protran; Schleicher & Schuell, Dassel, Germany) and probed with rabbit polyclonal antibodies against endostatin (HES.6) [ ] and human collagen XVIII (anti-all huXVIII QH4818 and anti-long huXVIII, QH1415.7) [ ], all used at a concentration 1 μg/ml in 5% fat-free milk powder in 1 × PBS, followed by a horseradish peroxidase-conjugated goat anti-rabbit antibody (Bio-Rad, Abingdon, UK).

Techniques: Western Blot

Journal: The Journal of pathology

Article Title: A 3D tri-culture system reveals that activin receptor-like kinase 5 and connective tissue growth factor drive human glomerulosclerosis

doi: 10.1002/path.4960

Figure Lengend Snippet: (A) MC 3D monoculture without the addition of TGFβ, and (B) in the presence of TGFβ, shown by H&E staining of paraffin-embedded sections. Human collagen type I/III (immunostained with an antibody that recognises both collagen type I and III) (C) and human collagen type IV (D) can be demonstrated in nodules by immunoperoxidase staining. (E) High-power view of MC nodule by scanning electron microscopy in cross section. (F) MC nodule count and collagen type I alpha1 (COL1a1), and collagen type IV alpha1 (COL4a1) RNA quantification (n=3). (G) Effect of ALK5 inhibition (Alk5i) on MC nodule formation (n=3). (H) Effect of SMAD2 or SMAD3 siRNA knockdown in MCs on nodule formation (n=3) with immunoblots demonstrating degree of knockdown. [DharmaFECT (DH), non-targeting siRNA control (siCP), siSMAD2 (siS2), siSMAD3 (siS3), total SMAD2 (tSMAD2), total SMAD3 (tSMAD3)]. Error bars represent 1 s.d. NS, not significant; ***, p<0.001; **, p<0.01: 2-tailed Student’s t test.

Article Snippet: After culture, matrices were embedded in HistoGel, fixed in 4% paraformaldehyde and paraffin wax-embedded for sectioning and staining for human collagen type IV (mouse anti-human collagen IV monoclonal antibody 2150–0121, Bio-Rad, CA, USA) and human collagen type I/III (rabbit anti-human collagen I/III polyclonal antibody 2150–2210, Bio-Rad, CA, USA).

Techniques: Staining, Immunoperoxidase Staining, Electron Microscopy, Inhibition, Western Blot