antibodies against trap  (Novus Biologicals)

Journal: Cardiovascular Research

Article Title: Bone morphogenetic protein 10 is increased in pre-capillary pulmonary hypertension patients

doi: 10.1093/cvr/cvaf028

Figure Lengend Snippet: Up-regulated local BMP10 expression in the right atrium of precPH patients. ( A ) Quantification of the relative BMP10 mRNA expression in control ( n = 9) and precPH ( n = 5 CTEPH) RA tissues. ( B and C ) Quantification of total RA BMP10 fluorescent area and RA cardiomyocytes BMP10 intensity levels in control ( n = 6) and precPH ( n = 4 PAH) paraffin-embedded RA tissue sections stained against BMP10, respectively. Representative immunofluorescent stainings of BMP10, Ulex-rhodamine (Ulex, endothelium), and cardiac TroponinT (cTnT, myocardium) in the negative control sample for anti-rabbit Alexa488 and anti-mouse Alexa647 ( D ), in the control ( E ), and in the precPH ( F ) RA tissues at 60×-oil magnification. ( D’–F’ ) Alexa488 single-channel images from the stainings in ( D–F ). ( E ′′ and F′′ ) Zoom-in images from ( E′ and F′ ) to appreciate the sarcomeric pattern of the BMP10 staining in the cardiomyocytes and the homogeneous staining in the vessels. Scale bars = 50 μm. Brightness and contrast for the Alexa488 channel have not been modified. Normality of data was checked and transformed if needed, and statistical differences between precPH patients and controls were tested using an independent sample t -test.

Article Snippet: Finally, we could not determine BMP10 activity directly using trap antibody against BMP10 (#MAB2926, R&D Systems), as described, because this antibody did not inhibit BMP10 transcriptional activity in our samples; therefore, we used the ALK1-Fc.

Techniques: Expressing, Control, Staining, Negative Control, Modification, Transformation Assay

Journal: Cardiovascular Research

Article Title: Bone morphogenetic protein 10 is increased in pre-capillary pulmonary hypertension patients

doi: 10.1093/cvr/cvaf028

Figure Lengend Snippet: Up-regulated local BMP10 activity in the right atrium of precPH patients. ( A ) Quantification of positive pSMAD1/5/8 nuclei in vascular and non-vascular cells within the RA tissues from precPH ( n = 4 PAH) and controls ( n = 5). ( B and C ) Representative immunofluorescent staining of positive pSMAD1/5/8 nuclei in vascular and non-vascular cells from control and precPH with rhodamine and Alexa488 single-channel images on the sides. ( D ) Quantification of positive ID3 nuclei in vascular and non-vascular cells within the RA tissues from precPH ( n = 4 PAH) and controls ( n = 5). ( E and F ) Representative immunofluorescent staining of positive ID3 nuclei in vascular and non-vascular cells from control and precPH with rhodamine and Alexa488 single-channel images on the sides. Arrowheads indicate positive pSMAD1/5/8 and ID3 nuclei. Zoom-in images are included within ( B , C , E , and F ). Nuclei were counterstained with Hoechst 33342 and vessels with Ulex-rhodamine ( B , C , E , and F ). Negative control images are shown in , . Scale bars = 50 μm. Vascular and non-vascular measurements are plotted with their own Y -axis on the left or right side, respectively. Normality of data was checked and transformed if needed, and statistical differences between precPH patients and controls were tested using a Wilcoxon rank-sum test (in A and D ).

Article Snippet: Finally, we could not determine BMP10 activity directly using trap antibody against BMP10 (#MAB2926, R&D Systems), as described, because this antibody did not inhibit BMP10 transcriptional activity in our samples; therefore, we used the ALK1-Fc.

Techniques: Activity Assay, Staining, Control, Negative Control, Transformation Assay

Journal: Cardiovascular Research

Article Title: Bone morphogenetic protein 10 is increased in pre-capillary pulmonary hypertension patients

doi: 10.1093/cvr/cvaf028

Figure Lengend Snippet: Higher BMP10 plasma levels in precPH patients compared with controls. ( A and B ) BMP10 protein circulating plasma levels in precPH patients ( n = 48) and subgroups ( n = 48: 22 iPAH, 14 hPAH, and 12 CTEPH), respectively, vs. controls ( n = 16). ( C and D ) BMP9 protein circulating plasma levels in precPH patients ( n = 45) and subgroups ( n = 45: 20 iPAH, 14 hPAH, and 11 CTEPH), respectively, vs. controls ( n = 16). ( E and F ) Correlation between BMP10 and BMP9 plasma levels in precPH patients ( n = 45) or subgroups ( n = 45: 20 iPAH, 14 hPAH, and 11 CTEPH), respectively, vs. controls ( n = 16). Logarithmic Y -axis is used in graphs ( A – D ). Data in ( A and B ) are y + 1 for logarithmic scale transformation. Normality of data was checked and transformed if needed. Statistical differences between precPH patients or precPH subgroups and controls were tested with an independent sample t -test or a one-way ANOVA, respectively. Associations were tested with univariate linear regression analysis.

Article Snippet: Finally, we could not determine BMP10 activity directly using trap antibody against BMP10 (#MAB2926, R&D Systems), as described, because this antibody did not inhibit BMP10 transcriptional activity in our samples; therefore, we used the ALK1-Fc.

Techniques: Clinical Proteomics, Transformation Assay

Journal: Cardiovascular Research

Article Title: Bone morphogenetic protein 10 is increased in pre-capillary pulmonary hypertension patients

doi: 10.1093/cvr/cvaf028

Figure Lengend Snippet: BMP10 transcriptional activity in precPH patients and controls. ( A ) Schematic explanation of the BRE-LUC reporter assay to determine BMP transcriptional activity in venous serum. Specific trap antibodies targeting BMP9 or BMP9 and BMP10 are used to assess BMP10 activity. Created with BioRender.com. B ) Relative BMP transcriptional activity as a luciferase read-out from the HMEC-BRE-LUC, endothelial cells expressing a BMP-specific luciferase reporter, in control ( n = 15) and precPH subgroups ( n = 21 iPAH, n = 13 hPAH, and n = 11 CTEPH) after incubation with phosphate-buffered saline (PBS) (baseline), anti-BMP9, or ALK1-Fc (inhibition of BMP9 and BMP10). ( C ) BMP10 activity in controls and precPH subgroups has been calculated from the subtraction of anti-BMP9 and ALK1-Fc to total BMP activity. Normality of data was checked and transformed if needed. Statistical differences between precPH patients and controls, and between baseline conditions and trap antibodies, were tested with an independent sample t -test or a one-way ANOVA, after which pairwise t -testing with Bonferroni correction was applied, respectively.

Article Snippet: Finally, we could not determine BMP10 activity directly using trap antibody against BMP10 (#MAB2926, R&D Systems), as described, because this antibody did not inhibit BMP10 transcriptional activity in our samples; therefore, we used the ALK1-Fc.

Techniques: Activity Assay, Reporter Assay, Luciferase, Expressing, Control, Incubation, Saline, Inhibition, Transformation Assay

Journal: Cardiovascular Research

Article Title: Bone morphogenetic protein 10 is increased in pre-capillary pulmonary hypertension patients

doi: 10.1093/cvr/cvaf028

Figure Lengend Snippet: Patients with more right atrial dilatation, reduced RV ejection fraction, and higher NT-proBNP have higher levels of circulation BMP10 activity. ( A and B ) BMP10 transcriptional activity in precPH patients with RA or RV dilation, respectively. ( C–E ) BMP10 transcriptional activity in precPH patients with high RAP, reduced RVEF, or high NT-proBNP, respectively. PrecPH patients were stratified according to RA volume (>79 mL/mm 2 for male patients or >69 mL/mm 2 for female patients), RV end-diastolic volume index (≥109 mL/mm 2 for males, and ≥97 mL/mm 2 for females), RAP (>14 mmHg), RVEF (<35%), and NT-proBNP levels (>1100 ng/L). Normality of data was checked and transformed if needed. Statistical differences between both groups were tested with an independent samples t -test.

Article Snippet: Finally, we could not determine BMP10 activity directly using trap antibody against BMP10 (#MAB2926, R&D Systems), as described, because this antibody did not inhibit BMP10 transcriptional activity in our samples; therefore, we used the ALK1-Fc.

Techniques: Activity Assay, Transformation Assay

Journal: Cardiovascular Research

Article Title: Bone morphogenetic protein 10 is increased in pre-capillary pulmonary hypertension patients

doi: 10.1093/cvr/cvaf028

Figure Lengend Snippet: Effect of pressure unloading on BMP10 activity in precPH patients. ( A ) Serum relative BMP activity at baseline and post-PEA in CTEPH patients. Incubation with anti-BMP9 only blocked BMP9 activity, while ALK1-Fc blocked both BMP9 and BMP10 activities. ( B ) Calculated BMP10 transcriptional activity at baseline and post-PEA ( n = 13), respectively. BMP10 transcriptional activity is calculated by subtracting BMP activity values after incubation with the trap antibodies. Normality of data was checked and transformed if needed. Statistical differences between baseline conditions and trap antibodies, and between baseline and post-PEA, were tested with an independent sample t -test.

Article Snippet: Finally, we could not determine BMP10 activity directly using trap antibody against BMP10 (#MAB2926, R&D Systems), as described, because this antibody did not inhibit BMP10 transcriptional activity in our samples; therefore, we used the ALK1-Fc.

Techniques: Activity Assay, Incubation, Transformation Assay

Journal: Cell Research

Article Title: Tau is a receptor with low affinity for glucocorticoids and is required for glucocorticoid-induced bone loss

doi: 10.1038/s41422-024-01016-0

Figure Lengend Snippet: a The schematic diagram of human proteome microarray, which contains over 20,000 individual proteins printed in duplicate, to identify binding partners of high-dose and low-dose dexamethasone, respectively. b , c Human proteome microarray analysis reveals the proteins (in blue) which bind to both low and high-dose dexamethasone, and proteins (in green) which selectively bind to high-dose dexamethasone. d Principle of DARTS assay for the isolation of proteins protected from degradation by dexamethasone. e Dexamethasone protects two groups of protein bands (highlighted in black rectangle) from degradation in DARTS using whole cell lysate from dexamethasone-treated Raw264.7 cells coupled with Coomassie blue staining. f Molecular weight (MW) plot of putative high-dose dexamethasone-binding proteins identified by human proteome microarray analysis. g The protective effects of serial doses of dexamethasone on Tau and GR from digestion by protease are evaluated by DARTS coupled with immunoblotting. GAPDH is resistant to protease under the condition and serves as a loading indicator. Representative image is shown ( n = 3). h Quantification of Tau and GR stability treated with serial dosages of dexamethasone assayed by DARTS ( n = 3). i The interaction between dexamethasone and Tau, assayed by solid phase binding. 10 mM Tau was coated to the plate, and a serial dilution of biotin-labeled dexamethasone was added, followed by incubation with HRP-labeled Streptavidin and its substrate ( n = 3). Inset shows the Scatchard plot analysis for K D value calculation. j – p One-step kinetic SPR assay for binding of Tau to different GCs, as indicated. q qRT-PCR analysis of Tau mRNA levels in different tissues, as indicated ( n = 3). r Double-immunoflurorescence staining of femur section using antibodies against Tau (green) and TRAP, osteocalcin (OCN) and sclerostin (SOST) (red). DAPI stains nuclei. Arrows indicate positive staining cells. Scale bar = 20 µm. BM, bone marrow. Data are means ± SD in h , i , q .

Article Snippet: Subsequently, sections were blocked with 10% goat serum for 30 min, and then incubated with primary antibodies against TRAP (1:100, PA5-116970, Invitrogen), osteocalcin (1:100, M173, Takara), sclerostin (1:100, PA5-46977, Invitrogen) or Tau (1:100, 10274-1-AP, ProteinTech) overnight at 4 °C.

Techniques: Microarray, Binding Assay, Isolation, Staining, Molecular Weight, Western Blot, Serial Dilution, Labeling, Incubation, SPR Assay, Quantitative RT-PCR

Journal: Cell Research

Article Title: Tau is a receptor with low affinity for glucocorticoids and is required for glucocorticoid-induced bone loss

doi: 10.1038/s41422-024-01016-0

Figure Lengend Snippet: a Immunoblotting of pTau S422, S396 and S202/T205 in Raw264.7 cells treated with low (L, 10 nM) or high (H, 10 µM) dose of dexamethasone along with 50 ng/mL RANKL for the indicated time. GAPDH is used as a loading control. Representative images from duplicate results are shown. b Immunoblotting of Tau in Tau knockout Raw264.7 cells transfected with empty vector, full length (FL) or Tau with serial point mutations. Representative images of duplicate results are shown. c , d Representative bright-field images ( c ) and corresponding quantification ( d ) of TRAP-positive multinuclear osteoclasts differentiated from FL- or Tau with serial point mutations- transfected Tau −/− Raw264.7 macrophage treated with 50 ng/mL RANKL and 10 µM dexamethasone for 5 days. Scale bar = 100 µm. e Confocal images of Raw264.7 cells labeled with antibodies for Tau and tubulin (left panel), and corresponding fluorescence signal intensity plots of Tau (red) and tubulin (green) vesicles (right panel). Cells are treated with or without 50 ng/mL RANKL and 10 µM dexamethasone. Arrows indicate regions in each cell where fluorescent signal intensity plots are obtained. f Confocal images of Raw264.7 cells labeled with antibodies for p-Tau Ser422 and tubulin (left panel), and corresponding fluorescence signal intensity plots of p-Tau Ser422 (red) and tubulin (green) vesicles (right panel). Cells are treated with or without 50 ng/mL RANKL and 10 µM dexamethasone. Arrows indicate regions in each cell where fluorescent signal intensity plots are obtained. g Experimental design and principle used to identify the potential kinases that are involved in dexamethasone-dependent p-Tau Ser422. h Summary of the potential kinases responsible for dexamethasone-dependent activation of Tau. i Knockdown efficiency of TTBK1 in Raw264.7 cells, measured by western blot ( n = 3). j Immunoblotting of dexamethasone-activated p-Tau Ser422 in Raw264.7 cells transfected with TTBK1 siRNA or treated with different kinase inhibitors, as indicated. k Densitometry analysis of immunoblotting results shown in j . l , m Representative bright-field images ( l ) and corresponding quantification ( m ) of TRAP-positive multinuclear osteoclast differentiated from Raw264.7 macrophages, which are transfected with TTBK1 siRNA or treated with different kinase inhibitor, followed by stimulating with 50 ng/mL RANKL and 10 µM dexamethasone for 5 days. Scale bar = 100 µm. n Confocal images of vehicle- or 10 µM dexamethasone-treated Raw264.7 cells labeled with antibodies for Tau and TTBK1 (left panel), and corresponding fluorescence signal intensity plots of Tau (red) and TTBK1 (green) vesicles (right panel). Arrows indicate regions in each cell where fluorescent signal intensity plots are obtained. o IP from 10 µM dexamethasone-treated Raw264.7 cells with anti-Tau antibody, and detection of TTBK1 by immunoblotting. Representative image of duplicate results is shown. p IP from 10 µM dexamethasone-treated Raw264.7 cells transfected with FLAG-tagged FL Tau or Tau with serial deletion mutations using anti-FLAG M2 resins, and detection of TTBK1 by immunoblotting ( n = 3). Data are means ± SD. P values are calculated by one-way ANOVA with Bonferroni post-hoc test ( d , k , m ).

Article Snippet: Subsequently, sections were blocked with 10% goat serum for 30 min, and then incubated with primary antibodies against TRAP (1:100, PA5-116970, Invitrogen), osteocalcin (1:100, M173, Takara), sclerostin (1:100, PA5-46977, Invitrogen) or Tau (1:100, 10274-1-AP, ProteinTech) overnight at 4 °C.

Techniques: Western Blot, Control, Knock-Out, Transfection, Plasmid Preparation, Labeling, Fluorescence, Activation Assay, Knockdown

Journal: Cell Research

Article Title: Tau is a receptor with low affinity for glucocorticoids and is required for glucocorticoid-induced bone loss

doi: 10.1038/s41422-024-01016-0

Figure Lengend Snippet: a Schematic for application of biochemical co-purification and mass spectrometry approaches to screen the transcriptional factors recruited to activated Tau by high-dose dexamethasone. Raw264.7 cells transfected with FLAG or FLAG-tagged Tau are treated with or without 10 µM dexamethasone for 30 min, followed by precipitation with anti-FLAG M2 resins. b Summary of the identified transcriptional factors recruited to Tau in 10 µM dexamethasone-treated Raw264.7 cells. c Knockdown efficiency of CTCF in Raw264.7 cells, measured by immunoblotting. d , e Representative bright-field images of TRAP-positive multinuclear osteoclast differentiated from Raw264.7 macrophages treated with 50 ng/mL RANKL and 10 µM dexamethasone for 5 days. Raw264.7 cells are treated with andrographolide (AGL, p50 inhibitor) or transfected with CTCF siRNA before differentiating into osteoclasts. Scale bar = 100 µm. f – h Gene expression levels of osteoclast differentiation markers NFATc1 (following 3 days of differentiation), CTSK and CTR (following 5 days of differentiation) determined by qRT-PCR. Bone marrow-derived macrophage isolated from WT, Tau −/− and GR −/− mice are differentiated with 20 ng/mL M-CSF and 50 ng/mL RANKL supplemented with low or high dose of dexamethasone ( n = 3). i IP from 10 µM dexamethasone-treated Raw264.7 cells with anti-Tau antibody, and detection of p105/p50 by immunoblotting. Representative image of duplicate results is shown. j IP from 10 µM dexamethasone-treated Raw264.7 cells transfected with FLAG-tagged FL or serial deletion mutations of Tau with anti-FLAG M2 resins, and detection of p105/p50 by immunoblotting. k , l Immunoblotting analysis ( k ) and quantification ( l ) of relative levels of p105 and p50 in control, Tau −/− and GR −/− Raw264.7 cells after 10 µM dexamethasone stimulation ( n = 3). m , n Immunoblotting analysis ( m ) and quantification ( n ) of relative levels of p105 and p50 in TTBK1 siRNA-transfected Raw264.7 cells after 10 µM dexamethasone stimulation. o , p Confocal images of Raw264.7 cells labeled with antibodies for p105/p50 ( o ), and relative fluorescence intensity of p105/p50 in cytoplasm and nuclear are shown ( p ) ( n = 3 biological replicates). q , r ChIP-qPCR assay of p50 in the NFATc1 promoter in WT ( q ) or GR −/− ( r ) Raw264.7 cells treated with 50 ng/mL RANKL for 1 h. s ChIP-qPCR assay of p50 in the NFATc1 promoter in Tau −/− Raw264.7 cells transfected with or without FL Tau or Tau S422P mutant followed by treatment with 50 ng/mL RANKL for 1 h. t , u ChIP-qPCR assay of p50 ( t ) and p65 ( u ) in the NFATc1 promoter in control or p50 siRNA knockdown Raw264.7 cell treated with RANKL in the presence or absence of Trx0237 for 1 h. Data are means ± SD. P values are calculated by one-way ANOVA with Bonferroni post-hoc test ( f – h , n , s – u ), two-way ANOVA with Bonferroni post-hoc test ( p ) and two-tailed unpaired Student’s t -test ( l , q , r ). n.s., not significant; ** P < 0.01.

Article Snippet: Subsequently, sections were blocked with 10% goat serum for 30 min, and then incubated with primary antibodies against TRAP (1:100, PA5-116970, Invitrogen), osteocalcin (1:100, M173, Takara), sclerostin (1:100, PA5-46977, Invitrogen) or Tau (1:100, 10274-1-AP, ProteinTech) overnight at 4 °C.

Techniques: Copurification, Mass Spectrometry, Transfection, Knockdown, Western Blot, Expressing, Quantitative RT-PCR, Derivative Assay, Isolation, Control, Labeling, Fluorescence, Mutagenesis, Two Tailed Test