Journal: Journal of Biological Chemistry

Article Title: Brorin, a Novel Secreted Bone Morphogenetic Protein Antagonist, Promotes Neurogenesis in Mouse Neural Precursor Cells

doi: 10.1074/jbc.m701570200

Figure Lengend Snippet: FIGURE 2. Detection of recombinant mouse Brorin protein. A, the lysate and culture medium of COS-7 cells expressing mouse Brorin cDNA were sep- arated by SDS-polyacrylamide gel electrophoresis under reducing condi- tions. Recombinant mouse Brorin protein with a Myc tag was detected by Western blotting with anti-Myc tag antibody. B, mouse Brorin cDNA was expressed in cultured High Five insect cells by infection with a recombinant baculovirus containing the mouse Brorin cDNA with a 3-terminal extension encoding Myc and His6 tags. Recombinant mouse Brorin protein was purified from the culture medium of High Five cells by affinity chromatography using Ni-NTA-agarose. Purified recombinant Brorin protein with bovine serum albumin as a carrier was separated by SDS-polyacrylamide gel electrophore- sis under reducing conditions. The protein was detected by protein staining with Coomassie Brilliant Blue R-250 (CBB) and by Western blotting with anti- Myc tag antibody (Western). Prestained protein markers were used as molec- ular mass standard proteins of 62.0, 47.5, and 32.5 kDa.

Article Snippet: Phosphorylation of Smad Protein in MC3T3-E1 Cells— MC3T3-E1 cells were plated in -MEM containing 10% FBS, 100 units/ml of penicillin G, and 100 g/ml of streptomycin at a density of 1 105 cells/well in 12-well plates for 48 h. Once confluent, the cells were cultured in -MEM containing 0.3% FBS, 100 units/ml of penicillin G, and 100 g/ml of streptomycin for 48 h. They were then cultured in -MEM containing 0.3% FBS, 100 units/ml of penicillin G, and 100 g/ml of streptomycin for 24 h and further cultured in -MEM containing 0.3% FBS, 100 units/ml of penicillin G, 100 g/ml of streptomy- 15844 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 282 • NUMBER 21 • MAY 25, 2007 at N Y U School of M edicine L ibrary on M arch 4, 2015 http://w w w .jbc.org/ D ow nloaded from cin, recombinant human BMP2 or BMP6 (0 or 10 ng/ml) (R&D Systems), recombinant mouse Noggin (0 or 100 ng/ml) (R&D Systems), and recombinant mouse Brorin protein (0 or 100 ng/ml) for 45 min.

Techniques: Recombinant, Expressing, Polyacrylamide Gel Electrophoresis, Western Blot, Cell Culture, Infection, Purification, Affinity Chromatography, Staining

Journal: Journal of Biological Chemistry

Article Title: Brorin, a Novel Secreted Bone Morphogenetic Protein Antagonist, Promotes Neurogenesis in Mouse Neural Precursor Cells

doi: 10.1074/jbc.m701570200

Figure Lengend Snippet: FIGURE 5. Effects of recombinant mouse Brorin protein on alkaline phosphatase activity and phosphorylation of Smad in MC3T3-E1 cells induced by BMP. A, MC3T3-E1 cells were treated with recombinant human BMP2 or BMP6 proteins and mouse Brorin or Noggin proteins for 72 h. After treatment, the alkaline phosphatase activity in MC3T3-E1 cells was measured. B, MC3T3-E1 cells were treated with recombinant human BMP2 or BMP6 proteins (0 or 10 ng/ml) and mouse Brorin or Noggin proteins (0 or 100 ng/ml) for 45 min. After treatment, phosphorylated Smad1/5/8 in MC3T3-E1 cells was detected by Western blotting.

Article Snippet: Phosphorylation of Smad Protein in MC3T3-E1 Cells— MC3T3-E1 cells were plated in -MEM containing 10% FBS, 100 units/ml of penicillin G, and 100 g/ml of streptomycin at a density of 1 105 cells/well in 12-well plates for 48 h. Once confluent, the cells were cultured in -MEM containing 0.3% FBS, 100 units/ml of penicillin G, and 100 g/ml of streptomycin for 48 h. They were then cultured in -MEM containing 0.3% FBS, 100 units/ml of penicillin G, and 100 g/ml of streptomycin for 24 h and further cultured in -MEM containing 0.3% FBS, 100 units/ml of penicillin G, 100 g/ml of streptomy- 15844 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 282 • NUMBER 21 • MAY 25, 2007 at N Y U School of M edicine L ibrary on M arch 4, 2015 http://w w w .jbc.org/ D ow nloaded from cin, recombinant human BMP2 or BMP6 (0 or 10 ng/ml) (R&D Systems), recombinant mouse Noggin (0 or 100 ng/ml) (R&D Systems), and recombinant mouse Brorin protein (0 or 100 ng/ml) for 45 min.

Techniques: Recombinant, Activity Assay, Phospho-proteomics, Western Blot

Journal: Journal of Biological Chemistry

Article Title: Brorin, a Novel Secreted Bone Morphogenetic Protein Antagonist, Promotes Neurogenesis in Mouse Neural Precursor Cells

doi: 10.1074/jbc.m701570200

Figure Lengend Snippet: FIGURE 6. Effects of recombinant mouse Brorin protein on neuronal and astrocytic differentiation in mouse neural precursor cells. A, mouse neu- ralprecursorcellswereculturedinthepresenceofrecombinantmouseBrorin protein (100 ng/ml) or FBS (10%). After being cultured for 3 days, the neural precursor cells were examined by double immunochemistry using anti-MAP2 and GFAP antibodies. Green and red signals indicate MAP2-positive and GFAP-positive cells, respectively. Blue signals indicate cell nuclei counter- stained with DAPI. Scale bar, 200 m. B, the effect of Brorin protein on the total number of cells was quantified by counting cell nuclei counter- stained with DAPI. C, the effect of Brorin protein on neuronal differentia- tion was quantified by counting MAP2-positive cells. D, the effect of Brorin protein on astrocytic differentiation was quantified by counting GFAP- positive cells. Results are means S.E. of four different fields from four independent slides. *, p 0.005.

Article Snippet: Phosphorylation of Smad Protein in MC3T3-E1 Cells— MC3T3-E1 cells were plated in -MEM containing 10% FBS, 100 units/ml of penicillin G, and 100 g/ml of streptomycin at a density of 1 105 cells/well in 12-well plates for 48 h. Once confluent, the cells were cultured in -MEM containing 0.3% FBS, 100 units/ml of penicillin G, and 100 g/ml of streptomycin for 48 h. They were then cultured in -MEM containing 0.3% FBS, 100 units/ml of penicillin G, and 100 g/ml of streptomycin for 24 h and further cultured in -MEM containing 0.3% FBS, 100 units/ml of penicillin G, 100 g/ml of streptomy- 15844 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 282 • NUMBER 21 • MAY 25, 2007 at N Y U School of M edicine L ibrary on M arch 4, 2015 http://w w w .jbc.org/ D ow nloaded from cin, recombinant human BMP2 or BMP6 (0 or 10 ng/ml) (R&D Systems), recombinant mouse Noggin (0 or 100 ng/ml) (R&D Systems), and recombinant mouse Brorin protein (0 or 100 ng/ml) for 45 min.

Techniques: Recombinant, Cell Culture, Staining

Journal: Scientific Reports

Article Title: Human TNF-Luc reporter mouse: A new model to quantify inflammatory responses

doi: 10.1038/s41598-018-36969-x

Figure Lengend Snippet: Effect of anti-inflammatory IL-10 on luciferase activity in hTNF.LucBAC BMDMs. BMDMs were either unstimulated (dotted line) or stimulated with 10 ng/mL LPS, in the absence (black line) or in the presence of increasing levels of recombinant murine IL-10, and luciferase activity was monitored over time; IL-10 concentrations 0.01 (orange), 0.1 (blue), 1 (green) and 10 ng/mL (red) ( a ). The ED50 of IL-10 as determined by the dose-dependent inhibition of luciferase activity in stimulated hTNF.LucBAC BMDMs was 0.6 ng/mL ( b ). Soluble TNF detected in the medium 24 h after LPS stimulation in the absence or presence of IL-10 ( c ). Correlation of IL-10 blockade of LPS-induced luciferase activity represented as area under the curve (AUC) with secreted mouse TNF ( d ). IKK-2 inhibitor BI605906 (BI) was used as a positive control for inhibition of luciferase activity and soluble TNF release. BMDMs left unstimulated (dotted line) or treated with 10 ng/mL of LPS, in the absence (black line) or presence of BI inhibitor at 0.01 μM (orange), 0.1 μM (blue), 1 μM (green) and 10 μM (red) ( e ). Luciferase activity (AUC) in the absence or presence of BI in LPS-induced cells (f ). Data are representative of three independent experiments, with triplicate cultures per experiment (N = 3, n = 3) and bars represent standard error of the mean.

Article Snippet: The crypt pellet was resuspended in ice-cold Matrigel (Scientific Laboratory Supplies, UK) (500 crypts/50 μL Matrigel) containing 50 mg/mL recombinant mouse epidermal growth factor (EGF), 100 ng/mL recombinant mouse Noggin CF and 500 ng/mL recombinant mouse R-spondin 1; all obtained from R&D Systems UK.

Techniques: Luciferase, Activity Assay, Recombinant, Inhibition, Positive Control