Journal: bioRxiv

Article Title: WFIKKN2 is a bifunctional axon guidance cue that signals through divergent DCC family receptors

doi: 10.1101/2023.06.15.544950

Figure Lengend Snippet: (A) Domain structure of murine (m)DCC family receptors and Netrin-1, drawn to scale. TM, transmembrane domain. (B) Brachial spinal cord transverse sections from E10.5, E11.5, and E12.5 mouse embryos were used for chromogenic in situ hybridization to detect Punc, Nope, and Prtg mRNA. All three genes show strong expression in DRGs (arrowheads) and the ventral horn (arrows) beginning at E11.5, with expression of Punc in ventral horn slightly delayed. Punc is also expressed in the progenitor zone (asterisk) at E10.5; all three receptors are expressed in the progenitor zone at E11.5 (C) An extracellular protein microarray was screened using Punc-ECD-Fc, Nope-ECD-Fc, and Prtg-ECD-His 6 as baits, revealing that all three receptors bind WFIKKN2. Black dots represent results for individual prey proteins from duplicate screening (arrays 1, 2). (D) Domain structure of mWFIKKN2. (E) Interactions of DCC family receptors with Netrin-1 and WFIKKN2 were examined in a COS-7-based binding assay. Netrin-1-AP specifically binds cells expressing DCC or Neo1, but not cells expressing Punc, Nope, or Prtg. Conversely, WFIKKN2-AP binds Punc-, Nope-, or Prtg-expressing cells, but not cells expressing DCC or Neo1. (F) WFIKKN2-Punc/Nope/Prtg binding was studied by SPR. Shown are SPR sensograms and Langmuir isotherms of steady-state responses fitted to a specific binding model for mWFIKKN2 as ligand on sensor chips with mPunc, mNope, or mPrtg ECDs as analytes at indicated concentrations. K D values are indicated, including standard error of the model fit. (G) Brachial-level transverse sections of E10.5, E11.5, and E12.5 embryos were used for RNAScope fluorescent in situ hybridization to detect WFIKKN2 mRNA. WFIKKN2 is expressed in tissue (yellow brackets) surrounding the spinal cord and DRG (red and orange dotted outlines, respectively) from E11.5 onwards. Asterisks indicate non-specific labeling from blood vessels. Scale bars, (B) 200 μm; (E) 50 μm; (G) 100 μm. Error bars indicate SEM.

Article Snippet: For the protein interaction screen, mPunc-ECD-Fc, a fusion of mNope ECD (G22-H953) to human Fc (Biotechne R&D Systems 1394-NP), and a fusion of mPrtg ECD (F36-A952) to a His 6 tag (Biotechne R&D Systems 6795-PR) were used as baits.

Techniques: Chromogenic In Situ Hybridization, Expressing, Microarray, Binding Assay, In Situ Hybridization, Labeling

Journal: PLoS ONE

Article Title: Role of integrin alpha8 in murine model of lung fibrosis

doi: 10.1371/journal.pone.0197937

Figure Lengend Snippet: (A) Western blot showing the absence of ITGA8 expression in Cre+ cells compared to Cre- cells. (B) Adhesion assay showing decreased binding to the substrates nephronectin (NPNT) and fibronectin (FN) by Cre+ cells. (C-G) PDGFRβ-selected cells were treated with TGFβ (10 ng/ml) or vehicle. mRNA expression is presented as fold change relative to Cre- in the untreated group (vehicle). Genes include: Itga8 (C), Col1a1 (D), Acta2 (E), Ctgf (F), and Fn1 (G). (* p <0.05, mean±SEM, n = 4).

Article Snippet: 96-well plates were coated with 1 μg/ml recombinant mouse nephronectin (R&D Systems) or rat plasma fibronectin (Sigma-Aldrich) at 4°C.

Techniques: Western Blot, Expressing, Cell Adhesion Assay, Binding Assay

Journal: Alzheimer's & dementia : the journal of the Alzheimer's Association

Article Title: Longitudinal CSF proteomics identifies NPTX2 as a prognostic biomarker of Alzheimer’s disease

doi: 10.1002/alz.12353

Figure Lengend Snippet: Longitudinal changes from baseline in cerebrospinal fluid concentration of neuronal pentraxin 2 (NPTX2) (raw data from individual participants: thin lines; mean changes resulting from the linear mixed effects (LME) model: thick lines, also shown in Figure 1 under “NPTX2”). A, Participants categorized as cognitively normal (CN) at baseline (blue); participants categorized as mild cognitive impairment (MCI) at baseline (red). B, Participants categorized as phosphorylated tau (p‐tau)181/amyloid beta (Aβ)1‐42 ratio positive at baseline (red); participants categorized as p‐tau181/Aβ1‐42 ratio negative at baseline (blue). C, Participants categorized as progressors (red); participants categorized as non‐progressors (blue)

Article Snippet: Due to the presence of endogenous levels of the five absolute quantitation target proteins in CSF, standard curve samples were prepared using recombinant proteins: CMGA (AbCam, #AB85486), FABPH (Sigma Aldrich, SRP4503), NPTX2 (R&D Systems, 7816‐NP‐050), SCG2 (LSBio, #LS‐ G25659 ), and VGF (Origene, TP309477) in 0.2 mg/mL bovine serum albumin in water.

Techniques: Concentration Assay

Journal: Alzheimer's & dementia : the journal of the Alzheimer's Association

Article Title: Longitudinal CSF proteomics identifies NPTX2 as a prognostic biomarker of Alzheimer’s disease

doi: 10.1002/alz.12353

Figure Lengend Snippet: Mean yearly rates of change in the CSF concentration of the five analytes under study resulting from the mixed‐effects modeling and their differences among various subsets of study participants

Article Snippet: Due to the presence of endogenous levels of the five absolute quantitation target proteins in CSF, standard curve samples were prepared using recombinant proteins: CMGA (AbCam, #AB85486), FABPH (Sigma Aldrich, SRP4503), NPTX2 (R&D Systems, 7816‐NP‐050), SCG2 (LSBio, #LS‐ G25659 ), and VGF (Origene, TP309477) in 0.2 mg/mL bovine serum albumin in water.

Techniques: Concentration Assay

Journal: Alzheimer's & dementia : the journal of the Alzheimer's Association

Article Title: Longitudinal CSF proteomics identifies NPTX2 as a prognostic biomarker of Alzheimer’s disease

doi: 10.1002/alz.12353

Figure Lengend Snippet: Correlations between neuronal pentraxin 2 (NPTX2) slopes and Mini‐Mental State Examination (MMSE), Alzheimer's Disease Assessment Scale–Cognitive 13‐item Subscale (ADAS‐Cog13) cognitive measures

Article Snippet: Due to the presence of endogenous levels of the five absolute quantitation target proteins in CSF, standard curve samples were prepared using recombinant proteins: CMGA (AbCam, #AB85486), FABPH (Sigma Aldrich, SRP4503), NPTX2 (R&D Systems, 7816‐NP‐050), SCG2 (LSBio, #LS‐ G25659 ), and VGF (Origene, TP309477) in 0.2 mg/mL bovine serum albumin in water.

Techniques:

Journal: eLife

Article Title: Nephronectin-integrin α8 signaling is required for proper migration of periocular neural crest cells during chick corneal development

doi: 10.7554/eLife.74307

Figure Lengend Snippet:

Article Snippet: Nunc Lab-tek II 8-well chamber slides (Sigma) were coated with 1.5 μg/cm 2 with poly- d -lysine (MP Biomedicals) for 1 hr at room temperature, followed by recombinant mouse or human Npnt (R&D Systems) at 1.5 μg/cm 2 for 2 hr at 37°C.

Techniques: Transfection, Construct, shRNA, Virus, In Vitro, Recombinant, Plasmid Preparation, PCR Cloning, Staining

Journal: Molecular and Cellular Biology

Article Title: Sortilin Facilitates Signaling of Ciliary Neurotrophic Factor and Related Helical Type 1 Cytokines Targeting the gp130/Leukemia Inhibitory Factor Receptor β Heterodimer

doi: 10.1128/mcb.00274-10

Figure Lengend Snippet: FIG. 8. Sortilin binds and enhances the signaling of CLC/CLF-1 and neuropoietin. (A) SPR analysis of CLC/CLF-1 (left) and neuro- poietin (NP) (right) binding to immobilized s-sortilin. The responses obtained in both the absence and presence of NT-mediated inhibition are shown, and the estimated Kd values are indicated. (B) Binding of CLC/CLF-1 in the absence or presence of a 200-fold excess of the 13-residue peptide constituting the C terminus of CNTF. (C) STAT3 phosphorylation induced by CLC/CLF-1 (left) and neuropoietin (right). BA/F3 transfectants expressing the indicated combinations of receptors were stimulated for 15 min at 37°C with CLC/CLF-1 (40 nM) plus sCNTFR (16 nM) or neuropoietin (1 nM) prior to Western blotting of lysed cells.

Article Snippet: Recombinant human CLC/CLF-1, CT-1, OSM, sCNTFR , soluble LIFR (sLIFR ), recombinant mouse neuropoietin, and goat anti-CNTFR were purchased from R&D Systems (Minneapolis, MN).

Techniques: Binding Assay, Inhibition, Residue, Phospho-proteomics, Expressing, Western Blot