Journal: Matrix biology : journal of the International Society for Matrix Biology

Article Title: Mechanistic interrogation of mutation-independent disease modulators of RDEB identifies the small leucine-rich proteoglycan PRELP as a TGF-β antagonist and inhibitor of fibrosis.

doi: 10.1016/j.matbio.2022.06.007

Figure Lengend Snippet: Fig. 7. Enhancement of RDEB keratinocytes attachment to fibroblasts-derived ECM by PRELP in the absence of Col VII. (A-B) Trypsin-based cell detachment assay of confluent layers of RDEB keratinocytes cultured over fibroblast-derived ECM. (A) Adherent cells were stained with 0.2% crystal-violet, lysed with 1%SDS, and measured at 580 nm. Cell adhe- sion was expressed as a percentage relative to non-trypsin treated wells. Values represent mean §SD of two indepen- dent assays performed in triplicates. ns= not significant, ****p 0.0001. (B) Representative micrographs of adhered cells after 15 min treatment with trypsin/EDTA evidence a significantly increased detachment of RDEB keratinocytes cultured over S-RDEBF-derived ECM. Right panel shows GFP expression in attached cells (bar: 250 mm).

Article Snippet: Membranes were blocked with 5% non-fat milk powder in TBS for 1 h at room temperature (RT) and incubated overnight with antibodies against: collagen VII N-terminus (pAb-LH7.2, gift from A. Nystr€om, University of Freiburg, Germany [51]), PRELP (H00005549-B01P, Novus Biologicals), tenascin-C (MAB2138, R&D Systems), collagen I (ab138492, Abcam), p-Smad2/ 3 (#8828), Smad2/3 (#8685), bIG-H3 (#5601) from Cell Signaling Technology, periostin (sc-398631), and GAPDH (sc-47724) from Santa Cruz Biotechnology.

Techniques: Derivative Assay, Cell Culture, Staining, Expressing

Journal: Scientific Reports

Article Title: A novel biomarker of MMP-cleaved prolargin is elevated in patients with psoriatic arthritis

doi: 10.1038/s41598-020-70327-0

Figure Lengend Snippet: Specificity of the PROM assay. The assay is specific to its selection peptide (HDFSSDLENV) and does not recognize truncated (DFSSDLENV), elongated (FHDFSSDLENV), or recombinant human prolargin protein (Gln21-Ile382), nor non-sense peptides (KSVDQASSRK). A twofold dilution of the peptides were added starting from 8 ng/ml. The background signal was tested using a non-sense coating peptide (Biotin-KSVDQASSRK). The data is presented as relative light units (RLU) function of peptide concentration.

Article Snippet: The specificity study included a nonsense peptide (KSVDQASSRK), an elongated peptide (FHDFSSDLENV), a truncated peptide (DFSSDLENV), a CHO-derived human PRELP protein (Gln21-Ile382, R&D Systems, MN, USA) and a nonsense coater (Biotin-KSVDQASSRK), used for determination of cross-reactivity.

Techniques: Selection, Recombinant, Concentration Assay

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: PRELP Enhances Host Innate Immunity against the Respiratory Tract Pathogen Moraxella catarrhalis .

doi: 10.4049/jimmunol.1601319

Figure Lengend Snippet: FIGURE 1. Respiratory tract pathogens bind PRELP. (A) [125I]-PRELP (0.75 mg/ml) was incubated with major species of human bacterial pathogens, and bound PRELP was measured in a gamma counter. Binding of PRELP was presented as the ratio (%) of radioactivity in the bacterial pellet (bound) over the initial amount of added radioactivity. Streptococcus pneumoniae CCUG1350, Haemophilus influenzae NTHi3655, and Moraxella catarrhalis RH4 bound more PRELP than Porphyromonas gingivalis W50, Pseudomonas aeruginosa ATCC27853, Streptococcus agalactiae KR624, and Streptococcus pyogenes CCUG25571. Mean 6 SD of four individual experiments are shown. Statistical differences were calculated using a one-way ANOVA with Dunnett’s post hoc test in comparison with control without bacteria. (B) PRELP is present in human BAL fluid collected from four patients with pneumonia. Human BAL fluid samples were concentrated and analyzed by Western blot. His-tagged PRELP is recombinantly expressed as 58 kDa. Several fragments of PRELP were detected in all patient samples, with three major fragments being at sizes around 55, 20, and 8 kDa, respectively. The concentration of intact human PRELP (55 kDa) was measured based on a standard curve generated from serial dilutions of PRELP. *p , 0.05, **p , 0.01, ***p , 0.001. ns, not significant.

Article Snippet: Polyclonal sheep anti-human PRELP Abs were purchased from R&D Systems (AF6447).

Techniques: Incubation, Binding Assay, Radioactivity, Comparison, Control, Bacteria, Western Blot, Concentration Assay, Generated

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: PRELP Enhances Host Innate Immunity against the Respiratory Tract Pathogen Moraxella catarrhalis .

doi: 10.4049/jimmunol.1601319

Figure Lengend Snippet: FIGURE 2. PRELP is present in human lung tissue. The specificity of the staining was ascertained using HEK293 cells expressing PRELP (A) or lacking expression of PRELP (B). Tissue sections from two donors were then subjected to immunohistochemical stain- ing for PRELP. PRELP (brown color) was detected in alveolar fluid (thick arrows) and in alveolar macrophages/monocytes (thin arrows) (C and D), in the tunica adventitia of blood vessels (arrowheads) (E and F), and in the myofibroblasts (G and H). Lung tissue was also stained using only secondary Ab as controls (I and J). The images are representative of all examined sections.

Article Snippet: Polyclonal sheep anti-human PRELP Abs were purchased from R&D Systems (AF6447).

Techniques: Staining, Expressing, Immunohistochemical staining

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: PRELP Enhances Host Innate Immunity against the Respiratory Tract Pathogen Moraxella catarrhalis .

doi: 10.4049/jimmunol.1601319

Figure Lengend Snippet: FIGURE 3. PRELP interacts directly with UspA2/A2H of M. catarrhalis. [125I]-PRELP was incubated with M. catarrhalis mutants devoid of selected surface proteins UspA1, UspA2/A2H, and MID that interact with various host proteins. UspA2/A2H mutant showed significantly reduced binding of PRELP in both M. catarrhalis Bc5 (A) and RH4 (B). (C) [125I]-PRELP binds the immobilized recombinant UspA2/A2H. Recombinant variants of UspA2H representing different regions (D) were immobilized with equimolar concentrations onto a microtiter plate and incubated with [125I]-PRELP. The radio- activity of bound [125I]-PRELP (E) was measured with a gamma counter. Background [125I]-PRELP binding to BSA on the plate was subtracted from all samples. Error bars represent SD (n = 3). Mutant versus parental wild-type (WT) strain, one-way ANOVA with Dunnett’s post hoc test (A and B); UspA2/ A2H versus corresponding BSA control, two-way ANOVA with Bonferroni post hoc test (C); UspA2H fragments versus full-length protein (50–720 aa), one-way ANOVA with Dunnett’s post hoc test (E). *p , 0.05, ***p , 0.001. ns, not significant.

Article Snippet: Polyclonal sheep anti-human PRELP Abs were purchased from R&D Systems (AF6447).

Techniques: Incubation, Mutagenesis, Binding Assay, Recombinant, Activity Assay, Control

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: PRELP Enhances Host Innate Immunity against the Respiratory Tract Pathogen Moraxella catarrhalis .

doi: 10.4049/jimmunol.1601319

Figure Lengend Snippet: FIGURE 4. PRELP binds to M. catarrhalis clinical isolates and enhances serum killing of M. catarrhalis. (A) [125I]-PRELP binds to clinical isolates expressing UspA2/A2H. The highly diverse head domains of UspA2/A2H are classified into NTERs 2A, 2B, 2C, 2H, and nontypeable. All tested clinical strains bound PRELP at varying degrees. Average PRELP binding for UspA2/A2H were NTER 2A 42.5% (n = 22), NTER 2B 37.1% (n = 11), NTER 2C 21.9% (n = 2), NTER 2H 41.6% (n = 11), and nontypeable 44.2% (n = 3). PRELP enhances serum killing of M. catarrhalis RH4 (B) and clinical isolates (C). Bacterial survival in human serum was defined as the ratio (%) of the CFUs at 30 min to time 0. Data are mean 6 SD of three independent experiments. Serum treated by C5 inhibitor OmCI was used as serum control, and 50 mg/ml PAB was used as a negative protein control. Versus 0 mg/ml PRELP, one-way ANOVA with Dunnett’s post hoc test (B); PRELP versus PAB control, one-way ANOVA with Bonferroni post hoc test (C). *p , 0.05, **p , 0.01, ***p , 0.001. ns, not significant.

Article Snippet: Polyclonal sheep anti-human PRELP Abs were purchased from R&D Systems (AF6447).

Techniques: Expressing, Binding Assay, Control

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: PRELP Enhances Host Innate Immunity against the Respiratory Tract Pathogen Moraxella catarrhalis .

doi: 10.4049/jimmunol.1601319

Figure Lengend Snippet: FIGURE 5. PRELP increases deposition of C3b and MAC on M. catarrhalis and inhibits bacterial binding of C4BP. Deposition of C3b (A), MAC (B), or C4BP (C and D) on M. catarrhalis was analyzed using flow cytometry. (E) PRELP competitively inhibits C4BP binding to the immobilized recombinant UspA2/A2H. BSA was coated as a negative protein control. Data in all figures are as mean 6 SD of three independent experiments. Heat-inactivated (HI) serum, in which complement is not active, was used as serum control, and PAB was used as a negative protein control. Versus 0 mg/ml PRELP, one-way ANOVAwith Dunnett’s post hoc test (A–D); Versus 0 mg/ml PRELP for corresponding protein, two-way ANOVAwith Bonferroni post hoc test (E). **p , 0.01, ***p , 0.001. ns, not significant.

Article Snippet: Polyclonal sheep anti-human PRELP Abs were purchased from R&D Systems (AF6447).

Techniques: Binding Assay, Cytometry, Recombinant, Control

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: PRELP Enhances Host Innate Immunity against the Respiratory Tract Pathogen Moraxella catarrhalis .

doi: 10.4049/jimmunol.1601319

Figure Lengend Snippet: FIGURE 6. Bacterial survival and com- plement deposition in depleted sera. (A) Bacterial survival in 5% C4BP and C1q depleted sera (C4BP dpl and C1q dpl). Deposition of C4BP (B), C3b (C), and MAC (D) on M. catarrhalis was analyzed using flow cytometry. C4BP was replen- ished at the physiological concentration (0.2 mg/ml). For C1q dpl, C1q was replenished to yield a final concentration of 60 mg/ml. PRELP (50 mg/ml) was used in all assays, and PAB was used as a negative protein control. Data in all figures are as mean 6 SD of three independent experi- ments. and analysed using a one-way ANOVA with Bonferroni post hoc test. *p , 0.05, **p , 0.01, ***p , 0.001. ns, not significant.

Article Snippet: Polyclonal sheep anti-human PRELP Abs were purchased from R&D Systems (AF6447).

Techniques: Cytometry, Concentration Assay, Control

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: PRELP Enhances Host Innate Immunity against the Respiratory Tract Pathogen Moraxella catarrhalis .

doi: 10.4049/jimmunol.1601319

Figure Lengend Snippet: FIGURE 7. PRELP enhances phagocytic killing of serum-opsonized M. catarrhalis by human neutrophils. Neutrophils were incubated with serum-opsonized M. catarrhalis in the presence of PRELP at 37˚C, 5% CO2. After incubation, total viable bacteria were enumerated, and bacterial survival (A) was calculated by dividing CFUs at 30, 60, or 90 min by CFUs at time 0. For phagocytosis assays, neutrophils were incubated with CFSE-labeled, serum-opsonized bacteria at 37 and 4˚C simultaneously with rotation. A total of 10,000 cells were gated and analyzed by flow cytometry. Neutrophils pretreated with CytoD were used as control of phagocytosis inhibition. PRELP (50 mg/ml) was used in all assays, and BSA was used as a negative protein control. Phagocytosis was determined by a phagocytic index (B), as described in Materials and Methods. Data are presented as mean 6 SD of three independent experiments and analyzed using a two-way ANOVAwith Bonferroni post hoc tests comparing the different conditions to that without PRELP (w/o PRELP) (A and B). **p , 0.01, ***p , 0.001. ns, not significant.

Article Snippet: Polyclonal sheep anti-human PRELP Abs were purchased from R&D Systems (AF6447).

Techniques: Incubation, Bacteria, Labeling, Cytometry, Control, Inhibition

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: PRELP Enhances Host Innate Immunity against the Respiratory Tract Pathogen Moraxella catarrhalis .

doi: 10.4049/jimmunol.1601319

Figure Lengend Snippet: FIGURE 8. COMP prevents bacterial colonization of human lung epithelial cells. PRELP binds to human epithelial cell line A549. Epithelial cell monolayers were incubated with a series of PRELP concentrations (0–500 mg/ml) for 1 h at 37˚C, 5% CO2 in serum-free medium. Binding of PRELP was detected using flow cytometry (A) with polyclonal sheep anti-human PRELP Abs, followed by fluorescently labeled secondary Abs. Binding of PRELP to human epithelial cell line A549 is dose dependent (B). PRELP reduces bacterial adherence to (C) and invasion (D) of lung epithelial A549 cells (MOI 10). After a 3 h incubation of bacteria with epithelial cells, total viable adherent or intracellular bacteria were enumerated and estimated on agar plates. Data are presented as mean 6 SD of three independent experiments and analyzed using a one-way ANOVA with Dunnett’s post hoc test comparing the different conditions to that in the absence of PRELP (0 mg/ml PRELP). *p , 0.05, **p , 0.01, ***p , 0.001. ns, not significant.

Article Snippet: Polyclonal sheep anti-human PRELP Abs were purchased from R&D Systems (AF6447).

Techniques: Incubation, Binding Assay, Cytometry, Labeling, Bacteria

Journal:

Article Title: Spatial and Temporal Regulation of Gene Expression in the Mammalian Growth Plate

doi: 10.1016/j.bone.2010.01.373

Figure Lengend Snippet: Different zones of the growth plate cartilage were micro-dissected. (A) The relative expression of mRNA in each zone was measured by expression microarray (Affymetrix Rat Genome Array 230 2.0). The data was background corrected and normalized using the MAS5 statistical algorithm. (B) The relative expression of Sfrp5, Pcp4, Efemp1, Lrrc17, Pcdh17, and Prelp was verified using quantitative real-time PCR. RZ, resting zone; PZ, proliferative zone; HZ, hypertrophic zone.

Article Snippet: The following pre-designed assays (Applied Biosystems) were used: Pcp4, Rn00564515_m1; Sfrp5, Rn01766277_m1; Efemp1, Rn01434325_m1; Lrrc17, Rn01442518_m1; Pcdh17, Rn0166237_m1; Prelp, Rn00584526_m1; c-Fos, Rn02396759_m1; Hif1a, Rn01472834_m1; Atf3, Rn00563784_m1; Frzb, Rn01746979_m1; Fgl2, Rn00584935_m1; Matn1, Rn01519781_m1; Irx4, Rn01537358_m1; Syt8, Rn00584120_m1; Tm4sf1, Rn01494890_m1; and 18S ribosomal RNA (18S rRNA): 18S-4319413E.

Techniques: Expressing, Microarray, Real-time Polymerase Chain Reaction

Journal: PLoS ONE

Article Title: Cells respond to deletion of CAV1 by increasing synthesis of extracellular matrix

doi: 10.1371/journal.pone.0205306

Figure Lengend Snippet: List of genes significantly up-regulated in CAV1-/ - MEFS. Only protein-encoding genes where q < 0.05 are shown, the full RNA-seq dataset is contained in S1 File . N = 4 biological replicates, each individual replicate comprising analysis of RNA pooled from all embryos from four mice.

Article Snippet: Probes used were Mm00515713_m1 (Fnb2), Mm00446968_m1 (Hprt), Mm 99999915_g1 (GAPDH), Mm01129316_m1 (Caveolin1), Mm01176187_m1 (Adamts15), Mm 04212217_m1 (Fras1), Mm01294828_m1 (Prelp), Mm00556810_m1 (Col6a6), Mm00801666_g1 (Col1a1) and Mm01166176_m1 (Col28a1).

Techniques:

Journal: Brazilian Journal of Medical and Biological Research

Article Title: Increased expression of ID2 , PRELP and SMOC2 genes in patients with endometriosis

doi: 10.1590/1414-431X20175782

Figure Lengend Snippet: Expression levels of ID2 ( A ), PRELP ( B ) and SMOC2 ( C ) genes in the eutopic endometrium and endometriotic lesions (endometrioma, peritoneal lesions and endometriotic lesions) of women with endometriosis. Horizontal lines indicate medians and interquartile range.

Article Snippet: The probes and primers for the ID2 (Hs00747379_m1), PRELP (Hs00160431_m1) and SMOC2 (Hs00405777_m1) genes, and the reference genes for the reaction, GAPDH (glyceraldehyde-3phosphate dehydrogenase) (Hs99999905_ml) and ACTB (actin, beta) (Hs99999903_m1), were obtained using Assay-on-DemandTM Gene Expression Products (Applied Biosystems, UK).

Techniques: Expressing

Journal: Molecular Vision

Article Title: A microarray analysis of gene expression patterns during early phases of newt lens regeneration

doi:

Figure Lengend Snippet: List of annotated contigs found in cluster D with sufficient sequence length.

Article Snippet: Contig_302 , XP_418054.2 , PREDICTED: prolargin , Gallus gallus , 271 , 5e-86 , 791.

Techniques: Sequencing