rabbit antibodies against s100a3  (Santa Cruz Biotechnology)

Journal: The European Respiratory Journal

Article Title: An atypical pulmonary fibrosis is associated with co-inheritance of mutations in the calcium binding protein genes S100A3 and S100A13

doi: 10.1183/13993003.02041-2018

Figure Lengend Snippet: Clinical characteristics of patients affected with pulmonary fibrosis. FVC: forced vital capacity; FEV 1 : forced expiratory volume in 1 s; TLC: total lung capacity; CT: computed tomography. a) Pedigrees of all families with pulmonary fibrosis with subsequent genotype analyses. Arrows indicate the proband from each family. Circles: females; squares: males; white symbols: not included in the study; white symbols with genotype: unaffected; black symbols: pulmonary fibrosis affected; +: wild-type “C” allele of S100A3 /wild-type sequence of S100A13 ; −: mutant “T” allele of S100A3 (c.229C>T)/4 bp deletion of S100A13 (c.238–241delATTG). b) CT scans at initial presentation of the three patients (F1:IV-1, IV-2 and IV-3) showing central traction bronchiectasis (long arrow). The distribution of fibrotic changes was peribronchovascular. The periphery of the lungs was spared. Global volume loss was seen with retracting subpleural fat in the lateral portions of the fissures (short arrows). c) CT scans late in the disease course of patient F1:IV-2: upper chest axial view (left), lower chest axial view (middle) and coronal view (right). There was progression of the peribronchovascular fibrotic changes and volume loss. Patches of ground-glass densities were randomly distributed. d, e) High-resolution CT scans of patients d) F2:IV-7 and e) F2:IV-8 showing central traction bronchiectasis (long arrows). The distribution of fibrotic changes is peribronchovascular and central. The periphery of the lungs was spared. Global volume loss is seen evident by retracting subplural fat in the lateral portions of the fissures (short arrows). f) Pulmonary function tests of five patients and family two members heterozygous for both the p.R77C and p.I80Gfs*13 variants in S100A3 and S100A13 , respectively. g) Pathology of one affected patient (F1:IV-2): generalised interstitial inflammation with fibrosis. The inflammation mostly consists of lymphocytes in a background of moderate interstitial fibrosis. No advanced lung fibrosis with honeycombing is identified. No granulomas, microgranuloma or vasculitis are noted. Scale bar: left image 100 µm, other images 50 µm. h) Pathology of another affected patient (F2:IV-7): interstitial inflammation with fibrosis in a diffuse pattern with no temporal heterogeneity. Advanced fibrosis seems to be sparing the subpleural space. No granulomas, microgranuloma or vasculitis are noted. Extensive sampling did not reveal a usual interstitial pneumonia-like pattern. Scale bar: left image 100 µm, other images 50 µm.

Article Snippet: For Western blots, cells were lysed, separated on either 7.5% SDS-PAGE (Bio-Rad, Hercules, CA, USA) or 4–12% gradient SDS-PAGE and transferred onto PVDF membranes (Life Technologies, Carlsbad, CA, USA) or nitrocellulose membranes (Hybond ECL; Amersham, Little Chalfont, UK), and immunoblotted using primary rabbit antibodies against S100A3 (Santa Cruz Biotechnology), S100A13, matrix metalloproteinase (MMP) 2, MMP9, tissue inhibitor of MMP (TIMP)-1, actin (Santa Cruz Biotechnology) or glyceraldehyde 3-phosphate dehydrogenase (GAPDH; Cell Signaling; Danvers, MA, USA), followed by peroxidase-conjugated goat anti-rabbit IgG (Jackson ImmunoResearch, West Grove, PA, USA).

Techniques: Computed Tomography, Sequencing, Mutagenesis, Sampling

Journal: The European Respiratory Journal

Article Title: An atypical pulmonary fibrosis is associated with co-inheritance of mutations in the calcium binding protein genes S100A3 and S100A13

doi: 10.1183/13993003.02041-2018

Figure Lengend Snippet: Molecular analyses in pulmonary fibrosis of Families 1(A and B) and 2. a) A single run of homozygosity as a result of homozygosity mapping shared by all seven affected patients between rs10802117 and rs11808053 confirming linkage analysis. In addition, a total of 24 unaffected family members displayed no homozygosity for this region of interest. b) Linkage analysis using a total of 17 individuals (seven affected and 10 unaffected) from the two families resulting in a peak where the maximum multipoint parametric logarithm of the odds score (pLOD MPT) was 5.28, corresponding to chromosome 1p12-q21.3 on the x-axis. c, d) Sequence chromatograms indicating the wild-type, homozygous affected and heterozygous carrier forms of c) the C to T transition at position c.229 changing the arginine residue to cysteine at position 77 of the S100A3 protein (c.229C>T; p.R77C) and d) the c.238–241delATTG (p.I80Gfs*13) in S100A13 . Mutation name is based on the full-length S100A3 (NM_002960) and S100A13 (NM_001024210) transcripts.

Article Snippet: For Western blots, cells were lysed, separated on either 7.5% SDS-PAGE (Bio-Rad, Hercules, CA, USA) or 4–12% gradient SDS-PAGE and transferred onto PVDF membranes (Life Technologies, Carlsbad, CA, USA) or nitrocellulose membranes (Hybond ECL; Amersham, Little Chalfont, UK), and immunoblotted using primary rabbit antibodies against S100A3 (Santa Cruz Biotechnology), S100A13, matrix metalloproteinase (MMP) 2, MMP9, tissue inhibitor of MMP (TIMP)-1, actin (Santa Cruz Biotechnology) or glyceraldehyde 3-phosphate dehydrogenase (GAPDH; Cell Signaling; Danvers, MA, USA), followed by peroxidase-conjugated goat anti-rabbit IgG (Jackson ImmunoResearch, West Grove, PA, USA).

Techniques: Sequencing, Mutagenesis

Journal: The European Respiratory Journal

Article Title: An atypical pulmonary fibrosis is associated with co-inheritance of mutations in the calcium binding protein genes S100A3 and S100A13

doi: 10.1183/13993003.02041-2018

Figure Lengend Snippet: Effect of S100A3 and S100A13 mutations on protein expression. AU: arbitrary units; IPF: idiopathic pulmonary fibrosis; GAPDH: glyceraldehyde 3-phosphate dehydrogenase. a) Upper images show immunofluorescence micrographs demonstrating reduced expression of S100A3 and S100A13 proteins in lung tissue from a normal control and an affected family member (F1:IV-2). Scale bar: 10 µm. Magnified areas of the indicated portions are shown in the lower images. b) Relative protein expression of S100A3 and S100A13 in normal control and lung tissues from two independent patients shown together with relative protein expression in an IPF patient. Histograms are mean± sd intensity of multiple fields in the stained samples. c) Confocal fluorescence laser scanning micrographs showing the reduced expression of S100A3 and S100A13 proteins in skin fibroblasts isolated from patients compared with controls and the corresponding Western blots. Data are representative of three independent experiments with cells isolated from two patients and two controls. Scale bar: 20 µm. d) Relative expression of S100A3 and S100A13 mRNA in skin fibroblasts isolated from normal controls and patients. Data are representative of at least three independent experiments. p-values are indicated when appropriate.

Article Snippet: For Western blots, cells were lysed, separated on either 7.5% SDS-PAGE (Bio-Rad, Hercules, CA, USA) or 4–12% gradient SDS-PAGE and transferred onto PVDF membranes (Life Technologies, Carlsbad, CA, USA) or nitrocellulose membranes (Hybond ECL; Amersham, Little Chalfont, UK), and immunoblotted using primary rabbit antibodies against S100A3 (Santa Cruz Biotechnology), S100A13, matrix metalloproteinase (MMP) 2, MMP9, tissue inhibitor of MMP (TIMP)-1, actin (Santa Cruz Biotechnology) or glyceraldehyde 3-phosphate dehydrogenase (GAPDH; Cell Signaling; Danvers, MA, USA), followed by peroxidase-conjugated goat anti-rabbit IgG (Jackson ImmunoResearch, West Grove, PA, USA).

Techniques: Expressing, Immunofluorescence, Staining, Fluorescence, Isolation, Western Blot

Journal: The European Respiratory Journal

Article Title: An atypical pulmonary fibrosis is associated with co-inheritance of mutations in the calcium binding protein genes S100A3 and S100A13

doi: 10.1183/13993003.02041-2018

Figure Lengend Snippet: Effect of S100A3 and S100A13 mutations on intracellular calcium changes. a) Intracellular calcium changes following stimulation of cultured skin fibroblasts isolated from a healthy control or a patient. Cells were stimulated with bradykinin (50 µM) (arrow). b) The histograms show maximum response to bradykinin. Experiments were performed on live single cells using confocal laser scanning microscopy. Data are expressed as mean± sem (n=23 and 24 for control and patients cells, respectively). Data are expressed as normalised fluorescence intensity ratio (F/F 0 ) relative to the averaged three images obtained prior to the addition of the stimulus and are representative of three independent experiments. c, d) Fibroblast growth factor-2 (10 ng·mL −1 )-stimulated cells, with results presented similar to a) and b). Data are expressed as mean± sem (n=10 and 7 for control and patient cells, respectively). Data are representative of 116 and 102 cells used in eight and 12 independent experiments from patient and control fibroblasts, respectively. e) Relative maximum calcium response to ionomycin (2 µM) in skin fibroblasts from controls and patients. Data are expressed as mean± sem (n=28 and 17 for control and patient cells, respectively). f) Mitochondrial calcium changes following stimulation of skin fibroblasts isolated from a healthy control or a patient with bradykinin (50 µM). Arrow indicates addition of bradykinin. Experiments were performed in live single cells using confocal laser scanning microscopy. All data are representative of cells isolated from two patients from the two unrelated families and two controls. p-values are indicated.

Article Snippet: For Western blots, cells were lysed, separated on either 7.5% SDS-PAGE (Bio-Rad, Hercules, CA, USA) or 4–12% gradient SDS-PAGE and transferred onto PVDF membranes (Life Technologies, Carlsbad, CA, USA) or nitrocellulose membranes (Hybond ECL; Amersham, Little Chalfont, UK), and immunoblotted using primary rabbit antibodies against S100A3 (Santa Cruz Biotechnology), S100A13, matrix metalloproteinase (MMP) 2, MMP9, tissue inhibitor of MMP (TIMP)-1, actin (Santa Cruz Biotechnology) or glyceraldehyde 3-phosphate dehydrogenase (GAPDH; Cell Signaling; Danvers, MA, USA), followed by peroxidase-conjugated goat anti-rabbit IgG (Jackson ImmunoResearch, West Grove, PA, USA).

Techniques: Cell Culture, Isolation, Confocal Laser Scanning Microscopy, Fluorescence

Journal: The European Respiratory Journal

Article Title: An atypical pulmonary fibrosis is associated with co-inheritance of mutations in the calcium binding protein genes S100A3 and S100A13

doi: 10.1183/13993003.02041-2018

Figure Lengend Snippet: Effect of S100A3 and S100A13 mutations on mitochondria. FITC: fluorescein isothiocyanate. a) Confocal fluorescence micrographs of isolated skin fibroblasts labelled with MitoTracker Red CMXRos (1 µM) and the corresponding three-dimensional intensity maps colour coded so that warm colours indicate high intensity and cold colours indicate low intensity. Scale bar: 20 µm. b) Flow cytometry of skin fibroblasts isolated from patient and control cells stained with MitoTracker Green FM. The inset shows mean± sem of fluorescence intensity in patients and control cells. Experiments were performed in triplicate and are representative of at least three independent experiments using 10 6 cells per sample. p-value is indicated. c) Transmission electron micrographs of cells isolated from healthy control and patient cells depicting differences in mitochondrial size (arrows) and loss of cristae. Scale bar: 1 µm. d) Effect of externally added oxidative insult (hydrogen peroxide 0.03%, arrow) on patient and control cells labelled with MitoTracker Red CMXRos. Data are representative of three independent experiments.

Article Snippet: For Western blots, cells were lysed, separated on either 7.5% SDS-PAGE (Bio-Rad, Hercules, CA, USA) or 4–12% gradient SDS-PAGE and transferred onto PVDF membranes (Life Technologies, Carlsbad, CA, USA) or nitrocellulose membranes (Hybond ECL; Amersham, Little Chalfont, UK), and immunoblotted using primary rabbit antibodies against S100A3 (Santa Cruz Biotechnology), S100A13, matrix metalloproteinase (MMP) 2, MMP9, tissue inhibitor of MMP (TIMP)-1, actin (Santa Cruz Biotechnology) or glyceraldehyde 3-phosphate dehydrogenase (GAPDH; Cell Signaling; Danvers, MA, USA), followed by peroxidase-conjugated goat anti-rabbit IgG (Jackson ImmunoResearch, West Grove, PA, USA).

Techniques: Fluorescence, Isolation, Flow Cytometry, Staining, Transmission Assay

Journal: The European Respiratory Journal

Article Title: An atypical pulmonary fibrosis is associated with co-inheritance of mutations in the calcium binding protein genes S100A3 and S100A13

doi: 10.1183/13993003.02041-2018

Figure Lengend Snippet: Effect of S100A3 and S100A13 mutations on extracellular matrix (ECM) components. MMP: matrix metalloproteinase; TIMP-1: tissue inhibitor of MMP-1; COL6A1: collagen α-1(VI) chain; COL1A2: collagen α-2(I) chain; CTHRC1: collagen triple helix repeat-containing protein 1; COL8A1: collagen α-1(VIII) chain; COL6A2: collagen α-2(VI) chain; PLOD1: procollagen-lysine, 2-oxoglutarate 5-dioxygenase 1. a) Western blots of MMP2, MMP9 and TIMP-1 expression by skin fibroblasts isolated from healthy controls and patients. Relative expression is depicted in the accompanying histograms. b, c) Differential expression of b) matrixins MMP1, MMP3 and MMP14, and c) ECM-associated proteins COL6A1, COL1A2, CTHRC1, COL8A1, COL6A2 and PLOD1. Normalised protein abundance of significantly differentially expressed proteins between patient and control samples is shown (fold change >1.5 and false discovery rate ∼3%). Yeast alcohol dehydrogenase standard (P00330) at a concentration of 200 fmol per injection was used for “Hi3” absolute quantifications of all identified proteins. The histogram bars correspond to the average protein expression between the two sample groups using the label-free liquid chromatography-mass spectrometry expression analysis system on the Progenesis QI for Proteomics platform. Data are expressed as mean± sem (n=3). p-values are indicated.

Article Snippet: For Western blots, cells were lysed, separated on either 7.5% SDS-PAGE (Bio-Rad, Hercules, CA, USA) or 4–12% gradient SDS-PAGE and transferred onto PVDF membranes (Life Technologies, Carlsbad, CA, USA) or nitrocellulose membranes (Hybond ECL; Amersham, Little Chalfont, UK), and immunoblotted using primary rabbit antibodies against S100A3 (Santa Cruz Biotechnology), S100A13, matrix metalloproteinase (MMP) 2, MMP9, tissue inhibitor of MMP (TIMP)-1, actin (Santa Cruz Biotechnology) or glyceraldehyde 3-phosphate dehydrogenase (GAPDH; Cell Signaling; Danvers, MA, USA), followed by peroxidase-conjugated goat anti-rabbit IgG (Jackson ImmunoResearch, West Grove, PA, USA).

Techniques: Western Blot, Expressing, Isolation, Concentration Assay, Injection, Liquid Chromatography, Mass Spectrometry