Journal: Development (Cambridge, England)

Article Title: A novel chordin-like BMP inhibitor, CHL2, expressed preferentially in chondrocytes of developing cartilage and osteoarthritic joint cartilage.

doi: 10.1242/dev.00901

Figure Lengend Snippet: Fig. 2. Direct interaction of mCHL2 with BMPs, and inhibition of BMP4 binding to BMP receptor ectodomain by mCHL2. (A) FLAG-tagged CHL2 protein. Proteins in the peak eluate from the hydroxyapatite column chromatography were separated by SDS-polyacrylamide gel electrophoresis under reducing conditions and then silver stained (Sambrook et al., 1989). The mCHL2-FLAG band was excised and the NH2- terminal amino acid sequence (vertical arrow in Fig. 1B) determined. (B) Immunoprecipitation/western blot analysis of mCHL2-FLAG individually mixed with BMP2 (a), BMP4 (b), BMP5 (c), BMP6 (d), BMP7 (e), GDF5 (f), activin A (g), TGFβ1 (h), TGFβ2 (i) or TGFβ3 (j), followed by treatment with αCHL2-COOH (lanes underlined). Immunocomplexes were detected using the corresponding antibodies (upper panels). Reactions only with mCHL2-FLAG, BMP, GDF, activin or TGFβ were also performed as negative controls. Each blot was further developed with M2 to confirm the presence of precipitated mCHL2-FLAG (lower panels). The TGFβ immunocomplexes (h-j) were separated into two sets; one was loaded on a non-reducing gel to visualize TGFβ (upper panels), and the other on a reducing gel to detect CHL2 (lower panels). Lanes not underlined were directly loaded with the indicated amount (ng) of mCHL2-FLAG, BMP, GDF, activin or TGFβ (for standards). (C) Inhibition of BMP4 binding to BMPR1B ectodomain by mCHL2-FLAG. The indicated amount of mCHL2-FLAG was first mixed with or without BMP4, and then BMPR1B-Fc or IgG-Fc was added. Protein complexes containing BMPR1B-Fc or IgG-Fc were selectively precipitated with protein A and subjected to western blot analysis (lanes underlined). Upper panel: bound BMP4 visualized with anti-BMP4 antibody. Middle panel: co-precipitation of mCHL2-FLAG checked with M2. Lower panel: precipitation of BMPR1B-Fc/IgG-Fc confirmed with anti-IgG-Fc antibody. For the standards, 0.04 µg of mCHL2-FLAG and 0.04 µg of BMP4 were loaded directly.

Article Snippet: Mouse chordin (mCHD-His); human BMP2, BMP7 and TGFβ1 (and mouse monoclonal antibodies against them); mouse monoclonal anti-human TGFβ3; mouse growth and differentiation factor 5 (GDF5); and affinity-purified goat polyclonal anti-mouse GDF5 were from R&D Systems (Minneapolis, MN).

Techniques: Inhibition, Binding Assay, Column Chromatography, Polyacrylamide Gel Electrophoresis, Staining, Sequencing, Immunoprecipitation, Western Blot

Journal: PLoS ONE

Article Title: Exploiting Ligand-Protein Conjugates to Monitor Ligand-Receptor Interactions

doi: 10.1371/journal.pone.0037598

Figure Lengend Snippet: (A) Scheme of the pulldown assay. The BG-ligand is immobilized on glutathione sepharose beads via GST-SNAP. Bound receptor proteins are concentrated on the beads after incubation with the extract and washing. (B) SNAP-eDHFR WT labeled with BG-647 (1 µM) was subjected to pulldown assay in the absence or presence of 100 µM NADPH using MTX immobilized on beads (MTX-BG +) or mock beads (MTX-BG -). Bound proteins were eluted with glutathione, submitted to SDS-PAGE and detected by in-gel fluorescence scanning. (C) Fluorescence signal of bound proteins normalized with the input signal in each gel (Mean±SD, n = 3). # represents P = 0.03 in paired t-test.

Article Snippet: The extract was subjected to affinity chromatography using Strep-tactin sepharose column (IBA) according to the manufacturer’s instructions.

Techniques: Incubation, Labeling, SDS Page, Fluorescence

Journal: Cell reports

Article Title: Ligands selectively tune the local and global motions of neurotensin receptor 1 (NTS 1 )

doi: 10.1016/j.celrep.2023.112015

Figure Lengend Snippet:

Article Snippet: Phusion HF DNA polymerase , NEB , Cat# M0530S.

Techniques: Virus, Recombinant, Functional Assay, Protease Inhibitor, Produced, Plasmid Preparation, Software, Electroporation, Fluorescence, Flow Cytometry, Chromatography, Affinity Column, Size-exclusion Chromatography, Purification

Journal: Journal of Toxicology

Article Title: Modified Rice Straw as Adsorbent Material to Remove Aflatoxin B 1 from Aqueous Media and as a Fiber Source in Fino Bread

doi: 10.1155/2016/6869582

Figure Lengend Snippet: HPLC chromatogram of AFB 1 (5 ng/mL) after incubation with different percentages of MRS at 25°C for 30 min.

Article Snippet: Immune affinity column AflaTest TM HPLC was obtained from VICAM (Watertown, MA, USA).

Techniques: Incubation

Journal: Journal of Toxicology

Article Title: Modified Rice Straw as Adsorbent Material to Remove Aflatoxin B 1 from Aqueous Media and as a Fiber Source in Fino Bread

doi: 10.1155/2016/6869582

Figure Lengend Snippet: It shows the HPLC chromatogram of AFB 1 (5 ng/mL) after incubation with MRS at different pH values.

Article Snippet: Immune affinity column AflaTest TM HPLC was obtained from VICAM (Watertown, MA, USA).

Techniques: Incubation