Journal: The Journal of biological chemistry

Article Title: HtrA2 regulates beta-amyloid precursor protein (APP) metabolism through endoplasmic reticulum-associated degradation.

doi: 10.1074/jbc.M702951200

Figure Lengend Snippet: FIGURE 2. Proteolytic cleavage of APP by HtrA2. Purified recombinant APP751-V5/His (A–C), APP695-V5/His (C), nectin-1-V5/His (D), or BACE1-His (D) was incubated with rec-HtrA2 in different conditions: (A) increasing concentrations of HtrA2 at 42 °C (15 or 45 min), (B) 132 nM HtrA2 at 37 °C (60 min) in a series of increasing buffer pH, (C) APP751-V5/His or APP695-V5/His were incubated with HtrA2 at 37 °C, pH 7.0 (60 min). D, 0.5 g of nectin-1-V5/His or BACE1-His were incubated with increasing concentrations of HtrA2 at 37 °C (60 min). West- ern blots were stained with APP C-terminal antibodies, V5 or His antibodies and subjected to densitometric analysis. The results are displayed as percentage of remaining APP holoprotein compared with the control samples containing no HtrA2.

Article Snippet: Antibodies, Peptides, and Recombinant Proteins—Recombinant human HtrA2 (rec-HtrA2), His-tagged recombinant BACE1 and HtrA2 antibody were from R&D Systems.

Techniques: Purification, Recombinant, Incubation, Staining, Control

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

Article Title: Immunoregulatory activity of the natural product laminarin varies widely as a result lt of its physical properties 1

doi: 10.4049/jimmunol.1701258

Figure Lengend Snippet: Saturation binding curves for rh and rm-Dectin-1 and two of the laminarins are shown. N=6 replicates for each laminarin and each receptor. At minimum a three log concentration range was employed with at least 10 data points for each analysis.

Article Snippet: Samples were analyzed for binding to recombinant human or mouse Dectin-1 (R&D Systems cat #1859-DC-050 and 1756-DC-050 respectively; both are C-terminal, extracellular truncated versions containing a 10 His N terminal tag) with an Octet Ks bio-layer interferometry instrument manufactured by ForteBio (Fremont CA).

Techniques: Binding Assay, Concentration Assay

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

Article Title: Immunoregulatory activity of the natural product laminarin varies widely as a result lt of its physical properties 1

doi: 10.4049/jimmunol.1701258

Figure Lengend Snippet: A. Dectin-1 in RAW or THP-1 cells was silenced using lentiviral particles containing shRNA targeted against the murine and human Clec7a gene. Control cells were also lentivirally transduced with a non-targeting shRNA against GFP. After antibiotic selection, single cell clones were propagated and screened for loss of Dectin-1 mRNA expression using qPCR (left) or protein expression (right). For protein analysis in RAWs, cells were pretreated with 30 μg/ml zymosan. Numbers under blots represent relative level of Dectin-1 protein vs. control cells as determined by quantification using the software ImageJ and normalization to actin protein levels. Dashed lines indicate image splicing to remove irrelevant lanes. B. TNFα production in response to 24 h treatment of the indicated cells, control (red) or Dectin-1 knockdown (black; clone 6 and clone 3 for RAW and THP-1, respectively), with increasing concentrations of indicated laminarin. Graphs are one representative experiment of at least three replicates.

Article Snippet: Samples were analyzed for binding to recombinant human or mouse Dectin-1 (R&D Systems cat #1859-DC-050 and 1756-DC-050 respectively; both are C-terminal, extracellular truncated versions containing a 10 His N terminal tag) with an Octet Ks bio-layer interferometry instrument manufactured by ForteBio (Fremont CA).

Techniques: shRNA, Transduction, Selection, Clone Assay, Expressing, Software