Journal: PLoS ONE

Article Title: Brain-specific angiogenesis inhibitor 1 is expressed in the Myo/Nog cell lineage

doi: 10.1371/journal.pone.0234792

Figure Lengend Snippet: Tissue sources and numbers of tissue and tissue sections screened by immunofluorescence localization of antibodies.

Article Snippet: The G8 IgM mAb and BAI1 IgG mAb (MAB4969, R&D Systems, Minneapolis, MN) were added in 100 μl of phosphate buffered saline (Applied Biosciences, ThermoFisher Scientific) at concentrations of 0.18–1.8 μg/well and 0.08–0.2 μg/100 μl/well, respectively.

Techniques: Immunofluorescence

Journal: PLoS ONE

Article Title: Brain-specific angiogenesis inhibitor 1 is expressed in the Myo/Nog cell lineage

doi: 10.1371/journal.pone.0234792

Figure Lengend Snippet: The G8 mAb was used to screen clones of HEK-239T cells expressing a single human membrane protein. G8 binding was detected by flow cytometry. A. Binding values were normalized and transformed to give a single numerical value for binding of the G8 mAb against each target protein (normalized target binding). Non-specific fluorescence was determined to be any value below three standard deviations above noise. G8 binding above background occurred with the clone expressing BAI1. B. Validation of binding of the G8 mAb to BAI1 or vector alone was carried out with HEK-293T cells transfected with the plasmid construct expressing target using a serial dilution of mAb. C. Binding of different concentrations of the G8 mAb was calculated as the signal to background ratio of fluorescence from the BAI1 expressing clone to empty vector clone.

Article Snippet: The G8 IgM mAb and BAI1 IgG mAb (MAB4969, R&D Systems, Minneapolis, MN) were added in 100 μl of phosphate buffered saline (Applied Biosciences, ThermoFisher Scientific) at concentrations of 0.18–1.8 μg/well and 0.08–0.2 μg/100 μl/well, respectively.

Techniques: Clone Assay, Expressing, Membrane, Binding Assay, Flow Cytometry, Transformation Assay, Fluorescence, Biomarker Discovery, Plasmid Preparation, Transfection, Construct, Serial Dilution

Journal: PLoS ONE

Article Title: Brain-specific angiogenesis inhibitor 1 is expressed in the Myo/Nog cell lineage

doi: 10.1371/journal.pone.0234792

Figure Lengend Snippet: Plates were coated with recombinant human BAI1 protein produced by CHO cells. Binding of the G8 IgM mAb and R&D BAI1 IgG mAb (a-BAI) was detected with anti-mouse IgM and IgG affinity purified F(ab’)2 secondary antibodies, respectively, conjugated with horseradish peroxidase. Both mAbs bound to BAI1 protein.

Article Snippet: The G8 IgM mAb and BAI1 IgG mAb (MAB4969, R&D Systems, Minneapolis, MN) were added in 100 μl of phosphate buffered saline (Applied Biosciences, ThermoFisher Scientific) at concentrations of 0.18–1.8 μg/well and 0.08–0.2 μg/100 μl/well, respectively.

Techniques: Recombinant, Produced, Binding Assay, Affinity Purification

Journal: PLoS ONE

Article Title: Brain-specific angiogenesis inhibitor 1 is expressed in the Myo/Nog cell lineage

doi: 10.1371/journal.pone.0234792

Figure Lengend Snippet: An alanine-scan library of BAI1 was constructed. The G8 and reference 5D6 mAbs were screened for binding to each individual BAI1 variant. A. Identification of candidate critical clones for G8 mAb binding was determined in duplicate by high throughput flow cytometry. For each point, background fluorescence was subtracted from the raw data that were then normalized to G8 reactivity with wild type BAI1. For each mutant clone, the mean binding value was plotted as a function of expression represented by control antibody reactivity. Candidate critical clones (red circles) were identified with a threshold (dashed line) of <40% binding of the G8 mAb and >50% of binding of the control 5D6 mAb to wild type BAI1. B. Identification of validated critical residues for G8 mAb binding. Candidate critical residues for G8 were rescreened in quadruplicate and the mean binding reactivities obtained. Validated critical residues for G8 binding (outlined in red) were residues whose mutations that resulted in <20% of wild type binding but positive (>70% binding) for binding of the 5D6 control mAb. Additional validated secondary residues (outlined in blue) were identified that did not meet the threshold guidelines but whose decreased binding activity and proximity to critical residues suggested that they may be part of the antibody epitope. C. Visualization of validated critical residues for G8 mAb binding. Critical residues (red spheres) and secondary residues (blue spheres) which may also contribute to binding were visualized on two Phyre-generated model structures that were combined to model BAI1 residues 262–934. The G8 critical residues were visualized on a model of the BAI1 thrombospondin repeat domains based on the structure of properdin (PDB ID# 1WOR; .

Article Snippet: The G8 IgM mAb and BAI1 IgG mAb (MAB4969, R&D Systems, Minneapolis, MN) were added in 100 μl of phosphate buffered saline (Applied Biosciences, ThermoFisher Scientific) at concentrations of 0.18–1.8 μg/well and 0.08–0.2 μg/100 μl/well, respectively.

Techniques: Construct, Binding Assay, Variant Assay, Clone Assay, High Throughput Screening Assay, Flow Cytometry, Fluorescence, Mutagenesis, Expressing, Control, Activity Assay, Generated

Journal: PLoS ONE

Article Title: Brain-specific angiogenesis inhibitor 1 is expressed in the Myo/Nog cell lineage

doi: 10.1371/journal.pone.0234792

Figure Lengend Snippet: Localization of antibodies to BAI1, Noggin and MyoD in the skin, eyes and brain. Human lens tissue and tissue sections of human tattooed skin, rabbit anterior cavity, rat retina and mouse brain were double labeled with the G8 and R&D BAI1 mAbs and antibodies to Noggin and MyoD. The total numbers of double labeled cells were counted in a subset of tissue sections. The total numbers of single labeled cells were quantified in all sections with the exception of MyoD-/R&D BAI1 mAb+ and MyoD-/G8- cells that were counted in a subset of skin and anterior cavity sections. The numbers of tissues and sections scored are indicated in parenthesis. The results are the mean ± standard deviation. The R&D BAI1 mAb co-localized with G8 and Noggin with rare exception. BAI1+ cells without MyoD were present in the skin and anterior cavity.

Article Snippet: The G8 IgM mAb and BAI1 IgG mAb (MAB4969, R&D Systems, Minneapolis, MN) were added in 100 μl of phosphate buffered saline (Applied Biosciences, ThermoFisher Scientific) at concentrations of 0.18–1.8 μg/well and 0.08–0.2 μg/100 μl/well, respectively.

Techniques: Labeling, Standard Deviation

Journal: PLoS ONE

Article Title: Brain-specific angiogenesis inhibitor 1 is expressed in the Myo/Nog cell lineage

doi: 10.1371/journal.pone.0234792

Figure Lengend Snippet: Tissue sections of human tattooed skin (A-C), human anterior lens tissue (D-F), rabbit eyes (G-I) and rat retina (J-L) were double labeled with the G8 (red) and BAI1 (green) mAbs. Nuclei were stained with Hoechst dye (blue). Unmerged images are shown in A, B, D, E and the insets in G-I and K and L. Overlap of red and green appear yellow in triple merged images in C, F, G-I, K and L. A merge of fluorescence and DIC is shown in the inset of C. The G8 and BAI1 mAbs labeled the same cells in the skin, human lens, rabbit lens (G), ciliary body (H) and cornea (I), and the inner plexiform and inner nuclear layers of the mouse retina (K and L). Minimal background fluorescence was observed in the anti-IgM and anti-IgG secondary antibody controls for the skin (M), human lens tissue (N), rabbit lens (O) and mouse retina P and Q. Bar = 9 μM in A-I and K-Q and 54 μM in J.

Article Snippet: The G8 IgM mAb and BAI1 IgG mAb (MAB4969, R&D Systems, Minneapolis, MN) were added in 100 μl of phosphate buffered saline (Applied Biosciences, ThermoFisher Scientific) at concentrations of 0.18–1.8 μg/well and 0.08–0.2 μg/100 μl/well, respectively.

Techniques: Labeling, Staining, Fluorescence

Journal: PLoS ONE

Article Title: Brain-specific angiogenesis inhibitor 1 is expressed in the Myo/Nog cell lineage

doi: 10.1371/journal.pone.0234792

Figure Lengend Snippet: Tissue sections of human tattooed skin (A and B), human anterior lens tissue (C) and rabbit anterior cavity (D-I) were double labeled with the R&D BAI1 mAb and antibodies to noggin or MyoD. The colors of the fluorescent secondary antibodies are indicated in each photograph. Overlap of red and green appears yellow in merged images. Nuclei were stained with Hoechst dye. Double labeled cells were present in the skin (A and B), anterior human lens tissue (C) and the rabbit lens (D and G), ciliary body (E and H) and cornea (F and I). Bar = 9 μM.

Article Snippet: The G8 IgM mAb and BAI1 IgG mAb (MAB4969, R&D Systems, Minneapolis, MN) were added in 100 μl of phosphate buffered saline (Applied Biosciences, ThermoFisher Scientific) at concentrations of 0.18–1.8 μg/well and 0.08–0.2 μg/100 μl/well, respectively.

Techniques: Labeling, Staining

Journal: PLoS ONE

Article Title: Brain-specific angiogenesis inhibitor 1 is expressed in the Myo/Nog cell lineage

doi: 10.1371/journal.pone.0234792

Figure Lengend Snippet: Tissue sections from the day-10 mouse brain were stained with hematoxylin and eosin (sagittal sections A and D; coronal sections B and C) or double labeled with the G8 and the R&D BAI1 mAbs, or G8 and antibodies to Iba1, NeuN or GFAP. The areas within the boxes of the H&E stained sections are shown at high magnification in the fluorescence photomicrographs. The colors of the fluorescent secondary antibodies are indicated in the unmerged photographs (E, F, H and I). Nuclei were stained with Hoechst dye. Overlap of red and green, when present, appears yellow in merged images (G, J, K, L and M). The G8 and BAI1 mAbs labeled the same subpopulation of cells in the hippocampal formation (box in A; E-G). The Noggin and BAI1 antibodies also bound to the same cells in the glomerular layer of the olfactory bulb (box in B; H-J). G8 did not co-localize with Iba1 (K, from box in A), NeuN (L from lower box in C) or GFAP (M from box in D). Minimal fluorescence was observed with the anti-IgM and anti-IgG (inset in G) or the anti-goat and anti-IgG (inset in J) secondary antibodies. Bar = 270 μM in A-D and 9 μM in E-M.

Article Snippet: The G8 IgM mAb and BAI1 IgG mAb (MAB4969, R&D Systems, Minneapolis, MN) were added in 100 μl of phosphate buffered saline (Applied Biosciences, ThermoFisher Scientific) at concentrations of 0.18–1.8 μg/well and 0.08–0.2 μg/100 μl/well, respectively.

Techniques: Staining, Labeling, Fluorescence

Journal: Nature communications

Article Title: Stabilin-2 modulates the efficiency of myoblast fusion during myogenic differentiation and muscle regeneration.

doi: 10.1038/ncomms10871

Figure Lengend Snippet: Figure 1 | Stabilin-2 expression in muscle tissues and during myogenic differentiation. (a) The expressions of PS-recognizing receptors (Tim1, Tim4, Bai1, stabilin-1 and stabilin-2) were examined in mouse skeletal muscles by quantitative real-time PCR. Data are presented as mean±s.d. of at least three independent experiments. (b,c) Expression levels of PS-recognizing receptors were examined in C2C12 cells (b) and primary myoblasts (c) during myogenic differentiation by quantitative real-time PCR. Data are presented as mean±s.d. of at least three independent experiments. GM, growth medium; DM, differentiation medium. (d,e) Expression levels of stabilin-2 protein were determined in C2C12 cells (d) and primary myoblasts (e) during myogenic differentiation by Western blotting. Representative results from three independent experiments are shown. MyHC, myosin heavy chain. Full-size blots are shown in Supplementary Fig. 15. (f,g) C2C12 cells (f) and primary myoblasts (g) were induced to differentiate in differentiation medium (DM), and the expressions of stabilin-2 and myosin heavy chain (MyHC) were analysed at the indicated times by immunostaining. Scale bar, 50 mm.

Article Snippet: Polyclonal anti-Bai1 antibody (NBP1–00723) was obtained from Novus Biologicals.

Techniques: Expressing, Muscles, Real-time Polymerase Chain Reaction, Western Blot, Immunostaining

Journal: Nature Communications

Article Title: Surfaceome dynamics reveal proteostasis-independent reorganization of neuronal surface proteins during development and synaptic plasticity

doi: 10.1038/s41467-020-18494-6

Figure Lengend Snippet: a cLTP paradigm b The plot shows averaged fast EPSC amplitudes for each cell (triangles) and their averages (mean ± s.e.m) for control (untreated, n = 45) and cLTP (glycine treated, n = 38) cells. Mann–Whitney test. c – f Boxplots of distributions of log2 surface abundances for replicates of cLTP ( n = 12) and control ( n = 12). Boxes indicate median and percentiles (25th and 75th). g Volcano plot of statistical significance ( y -axis) and surface abundance change ( x -axis). Horizontal line is located at an adjusted p -value of 0.05. h Proteins with significantly different surface abundances (fold-change > 1.5 and p < 0.05) after cLTP grouped into categories. Shape size indicates scaled −log10 adjusted p -value. Border color indicates fold-change directionality (green > 0, purple < 0). Edges represent string confidence > 0.7. i Representative images of analysed primary dendrites before and after cLTP induction. Bars 5 µm. j Quantification of mean fluorescent intensity of antibody signal on the primary dendrite surface using antibodies against GluA: n = 63, Bai1: n = 42, Adcy3: n = 28 (CTRL) n = 22 (cLTP) cells/group. Means + 95% CI presented. Two-way T -test (Adcy3 + Bai1) or Mann–Whitney test (GluA). * p < 0.05, ** p < 0.01, **** p < 0.0001. Source data are provided as a Source Data file.

Article Snippet: Antibodies targeting the extracellular domains of ADGRB1 (ABR-021, Alomone), AMPARs (182 411, Synaptic Systems), and AC3 (AAR-043, Alomone) were diluted 1:100 in blocking solution containing 10% normal goat serum (NGS) and incubated with the cells for 60 min.

Techniques: MANN-WHITNEY

Journal: Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie

Article Title: Tandem mass tag (TMT) proteomic analysis of fetal lungs revealed differential expression of tight junction proteins in a rat model of congenital diaphragmatic hernia.

doi: 10.1016/j.biopha.2019.109621

Figure Lengend Snippet: Fig. 6. Western blotting analysis of tight junction protein expression in the lungs of fetal rats. (A, B) Representative immunoblotting and densitometric analysis of tight junction protein expression in the fetal lung. Results were normalized relative to the expres- sion of β-actin (n = 6 per group, *P < 0.05, vs. con- trol). Western blot analysis showed increased levels of Cldn3 and decreased levels of Magi1 and Myh9 in the CDH fetal lungs.

Article Snippet: The membranes were blocked in 5% nonfat dry milk for 60min and then incubated with rabbit polyclonal anti-CLDN3 (Proteintech, Chicago, USA), rabbit polyclonal anti-MAGI1 antibody (Absin, Shanghai, China), rabbit polyclonal anti-MYH9 (Proteintech Group, Chicago, USA), and mouse monoclonal β-actin (Proteintech Group, Chicago, USA) overnight at 4 °C.

Techniques: Western Blot, Expressing

Journal: Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie

Article Title: Tandem mass tag (TMT) proteomic analysis of fetal lungs revealed differential expression of tight junction proteins in a rat model of congenital diaphragmatic hernia.

doi: 10.1016/j.biopha.2019.109621

Figure Lengend Snippet: Fig. 7. IHC analysis of tight-junction proteins in the lungs of fetal rats. (A) Representative photomicrographs of IHC-staining for Cldn3, Magi1, and Myh9 in the lung sections from CDH (Left panel) and Control (right panel) fetal rats. (Original magnification ×400, scale bar = 100 μm). Cldn3 and Magi1 were mainly expressed in the epithelial cells, and some weak staining was also found in the mesenchymal cells. Myh9 protein localized to both epithelial and mesenchymal cells. (B) Semi‑quantitative analysis of IHC staining (mean density). n = 3 per group, *P < 0.05, vs. control.

Article Snippet: The membranes were blocked in 5% nonfat dry milk for 60min and then incubated with rabbit polyclonal anti-CLDN3 (Proteintech, Chicago, USA), rabbit polyclonal anti-MAGI1 antibody (Absin, Shanghai, China), rabbit polyclonal anti-MYH9 (Proteintech Group, Chicago, USA), and mouse monoclonal β-actin (Proteintech Group, Chicago, USA) overnight at 4 °C.

Techniques: Immunohistochemistry, Control, Staining

Journal: Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie

Article Title: Tandem mass tag (TMT) proteomic analysis of fetal lungs revealed differential expression of tight junction proteins in a rat model of congenital diaphragmatic hernia.

doi: 10.1016/j.biopha.2019.109621

Figure Lengend Snippet: Fig. 8. Temporal expression of tight junction mRNA in CDH lungs. (A–C) Cldn3, Magi1, and Myh9 mRNA levels in CDH lungs were determined by quantitative RT- PCR at different time points. β-actin was used as a housekeeping gene. The results were normalized relative to the same-aged control group (n = 8 per group, *P < 0.05, vs. control at the same time point).

Article Snippet: The membranes were blocked in 5% nonfat dry milk for 60min and then incubated with rabbit polyclonal anti-CLDN3 (Proteintech, Chicago, USA), rabbit polyclonal anti-MAGI1 antibody (Absin, Shanghai, China), rabbit polyclonal anti-MYH9 (Proteintech Group, Chicago, USA), and mouse monoclonal β-actin (Proteintech Group, Chicago, USA) overnight at 4 °C.

Techniques: Expressing, Quantitative RT-PCR, Control