Journal: PLoS ONE

Article Title: BMP-7 induces apoptosis in human germinal center B cells and is influenced by TGF-β receptor type I ALK5

doi: 10.1371/journal.pone.0177188

Figure Lengend Snippet: (A-C) GC B cells were obtained by immunomagnetic bead separation and co-cultured with HK cells in the presence of CD40L/IL-21 with or without BMP-7 and apoptosis was measured by TUNEL assay. (A) Cells were cultured for up to 3 days and analyzed by flow cytometry. Shown is one representative of 2 donors. (B) After 2 days in culture, TUNEL staining (green) and Hoechst staining (blue) was detected by confocal microscopy. Representative images from one of three independent experiments are presented. Scale bar represents 30 μm. (C) The ALK 2/3 and ALK 4/5/7 selective inhibitors, LDN193189 and SB431542, respectively, were added to the cultures as specified and apoptosis was measured by flow cytometry at day 2. Mean ±SEM, n = 4, (D) Single cell suspensions from human tonsils were stained with lineage markers and anti-ALK4, anti-ALK5 or anti-ALK7 antibodies, and analyzed by flow cytometry. Shown are histogram overlays of receptor expression as compared to an irrelevant control. All experiments were repeated at least twice. Statistical testing was performed against CD40L/IL-21 condition. * p < 0.05; two-tailed, paired Student’s t -test.

Article Snippet: The following primary antibodies were used: biotinylated anti-ActRIIa (BAF340), -ActRIIb (BAF339), -BMPRII (BAF811),—ALK2 (BAF637), -ALK3 (BAF820), -ALK4 (BAF222) -ALK5 (BAF 3025), -ALK6 (BAF505), biotinylated goat IgG (BAF108) (R&D Systems, MN, USA).

Techniques: Cell Culture, TUNEL Assay, Flow Cytometry, Staining, Confocal Microscopy, Expressing, Control, Two Tailed Test

Journal: PLoS ONE

Article Title: BMP-7 induces apoptosis in human germinal center B cells and is influenced by TGF-β receptor type I ALK5

doi: 10.1371/journal.pone.0177188

Figure Lengend Snippet: Mino cells were transduced with truncated ALK5 or truncated ALK4. (A) The cells were stained with biotinylated anti-ALK5, followed by streptavidin PE or by biotinylated anti-ALK4, followed by streptavidin APC, and analyzed by flow cytometry. Receptor expression is compared in GFP + or mCherry + transduced cells vs. GFP - /mCherry - non-transduced cells. (B-C) The transduced Mino cells were cultured in serum free media (X-VIVO 15) over night and then left in medium alone (unstim) or stimulated with BMP-2, BMP-7 or TGF-β for 60 min, before detection of phosphorylated (p-) Smad 1/5 or p-Smad 2/3 by flow cytometry. (B) One representative experiment showing p-SMAD1/5 vs. GFP in truncated ALK5-2A-GFP expressing cells. (C) BMP- or TGF-β-induced phosphorylation is shown relative to unstimulated cells, using arcsinh transformation of median fluorescence intensity data. Mean ± SEM, n = 5. (D-E): Transduced Mino cells were cultured in X-VIVO 15 and left unstimulated or stimulated with TGF-β or BMP-7 for 72 hours and stained for active caspase-3 before analysis by flow cytometry. Shown here is active caspase-3 staining of control cells and transduced cells for (D) one representative experiment and (E) mean ± SEM, n = 3. * p < 0.05; two-tailed, paired Student’s t -test.

Article Snippet: The following primary antibodies were used: biotinylated anti-ActRIIa (BAF340), -ActRIIb (BAF339), -BMPRII (BAF811),—ALK2 (BAF637), -ALK3 (BAF820), -ALK4 (BAF222) -ALK5 (BAF 3025), -ALK6 (BAF505), biotinylated goat IgG (BAF108) (R&D Systems, MN, USA).

Techniques: Transduction, Staining, Flow Cytometry, Expressing, Cell Culture, Phospho-proteomics, Transformation Assay, Fluorescence, Control, Two Tailed Test

Journal: PLoS ONE

Article Title: BMP-7 induces apoptosis in human germinal center B cells and is influenced by TGF-β receptor type I ALK5

doi: 10.1371/journal.pone.0177188

Figure Lengend Snippet: Mino cells were transduced with truncated ALK5 or truncated ALK4. (A) The cells were stained with biotinylated anti-ALK5, followed by streptavidin PE or by biotinylated anti-ALK4, followed by streptavidin APC, and analyzed by flow cytometry. Receptor expression is compared in GFP + or mCherry + transduced cells vs. GFP - /mCherry - non-transduced cells. (B-C) The transduced Mino cells were cultured in serum free media (X-VIVO 15) over night and then left in medium alone (unstim) or stimulated with BMP-2, BMP-7 or TGF-β for 60 min, before detection of phosphorylated (p-) Smad 1/5 or p-Smad 2/3 by flow cytometry. (B) One representative experiment showing p-SMAD1/5 vs. GFP in truncated ALK5-2A-GFP expressing cells. (C) BMP- or TGF-β-induced phosphorylation is shown relative to unstimulated cells, using arcsinh transformation of median fluorescence intensity data. Mean ± SEM, n = 5. (D-E): Transduced Mino cells were cultured in X-VIVO 15 and left unstimulated or stimulated with TGF-β or BMP-7 for 72 hours and stained for active caspase-3 before analysis by flow cytometry. Shown here is active caspase-3 staining of control cells and transduced cells for (D) one representative experiment and (E) mean ± SEM, n = 3. * p < 0.05; two-tailed, paired Student’s t -test.

Article Snippet: The following primary antibodies were used: biotinylated anti-ActRIIa (BAF340), -ActRIIb (BAF339), -BMPRII (BAF811),—ALK2 (BAF637), -ALK3 (BAF820), -ALK4 (BAF222) -ALK5 (BAF 3025), -ALK6 (BAF505), biotinylated goat IgG (BAF108) (R&D Systems, MN, USA).

Techniques: Transduction, Staining, Flow Cytometry, Expressing, Cell Culture, Phospho-proteomics, Transformation Assay, Fluorescence, Control, Two Tailed Test

Journal: Drug testing and analysis

Article Title: Gel Electrophoretic Detection of Black Market ACE-031.

doi: 10.1002/dta.3898

Figure Lengend Snippet: FIGURE 3 | Separation characteristics of 14 ACE-031 black market products (BM01 to BM14) on SDS-PAGE (10% T, MOPS running buffer; ca. 10 μg on gel, Coomassie R-250 stain). Twelve products looked quite identical after electrophoretic separation (the green arrow indicates the main band). Product BM04 did not contain ACE-031 but black market follistatin 344 instead (red arrow; also see Figure 4B). No proteins were found in BM14. Product BM06 was chosen for the administration study. Mark12 is the molecular weight marker.

Article Snippet: The primary antibodies (custom- biotinylated human activin RIIB antibody clone 60,408 [MAB3392], biotinylated polyclonal human activin RIIB antibody [BAF339], anti- His- tag antibody clone AD1.1.10 [MAB050], biotinylated anti- follistatin antibody [BAF669]) were from R&D Systems (Minneapolis, MN) and the HRP- labelled goat anti- mouse IgG (H + L) highly cross- adsorbed secondary antibody from Thermo Fisher Scientific (31432).

Techniques: SDS Page, Staining, Molecular Weight, Marker

Journal: Drug testing and analysis

Article Title: Gel Electrophoretic Detection of Black Market ACE-031.

doi: 10.1002/dta.3898

Figure Lengend Snippet: FIGURE 4 | Separation characteristics of 14 ACE-031 black market products (BM01 to BM14) on SDS-PAGE (10% T, MOPS running buffer; ca. 50 ng on gel). Immunoblots with anti-ACVR2B antibody BAF339 (A), anti-follistatin antibody BAF669 (B), and anti-His antibody MAB050 (C). BM04 did not contain immunoreactive ACVR2B (A) bust instead BM follistatin 344 (B). All ACE-031 products except BM03 were positive for His-tags (C). Luspatercept (Lane 1) served as positive control for an ACVR2B-Fc fusion protein, and the two black market follistatins (BM FS344-1 and -2) as pos- itive controls for follistatins with His-tags (Lanes 16 and 17). The red arrow indicates the monomer, and the green indicates the dimer and trimer of the black market products.

Article Snippet: The primary antibodies (custom- biotinylated human activin RIIB antibody clone 60,408 [MAB3392], biotinylated polyclonal human activin RIIB antibody [BAF339], anti- His- tag antibody clone AD1.1.10 [MAB050], biotinylated anti- follistatin antibody [BAF669]) were from R&D Systems (Minneapolis, MN) and the HRP- labelled goat anti- mouse IgG (H + L) highly cross- adsorbed secondary antibody from Thermo Fisher Scientific (31432).

Techniques: SDS Page, Western Blot, Positive Control

Journal: Drug testing and analysis

Article Title: Gel Electrophoretic Detection of Black Market ACE-031.

doi: 10.1002/dta.3898

Figure Lengend Snippet: FIGURE 9 | Analysis of five female and five male rat serum samples 24/48/168 h after administration of BM ACE-031 (red arrow). IP with poly- clonal anti-ACVR2B antibody followed by SDS-PAGE and immunoblot with biotinylated clone 60408 anti-ACVR2B antibody and streptavidin-POD. Note the strong difference in signal intensity between the black market (red) product and the Reblozyl (blue), which indicates that the BM product contains a much lower amount of immunoreactive ACVR2B. NQC (negative rat serum), PQC (Reblozyl/BM ACE-031 positive rat serum). Lanes marked with a “+”,“+/−”, or “−” refer to samples in which BM ACE-031 was “clearly,” “not clearly,” or “not” identified.

Article Snippet: The primary antibodies (custom- biotinylated human activin RIIB antibody clone 60,408 [MAB3392], biotinylated polyclonal human activin RIIB antibody [BAF339], anti- His- tag antibody clone AD1.1.10 [MAB050], biotinylated anti- follistatin antibody [BAF669]) were from R&D Systems (Minneapolis, MN) and the HRP- labelled goat anti- mouse IgG (H + L) highly cross- adsorbed secondary antibody from Thermo Fisher Scientific (31432).

Techniques: SDS Page, Western Blot

Journal: Drug testing and analysis

Article Title: Gel Electrophoretic Detection of Black Market ACE-031.

doi: 10.1002/dta.3898

Figure Lengend Snippet: FIGURE 10 | In vitro study of BM ACE-031 using human and rat liver microsomes. SDS-PAGE and Western blot with biotinylated clone 60408 anti-ACVR2B antibody and streptavidin-POD.

Article Snippet: The primary antibodies (custom- biotinylated human activin RIIB antibody clone 60,408 [MAB3392], biotinylated polyclonal human activin RIIB antibody [BAF339], anti- His- tag antibody clone AD1.1.10 [MAB050], biotinylated anti- follistatin antibody [BAF669]) were from R&D Systems (Minneapolis, MN) and the HRP- labelled goat anti- mouse IgG (H + L) highly cross- adsorbed secondary antibody from Thermo Fisher Scientific (31432).

Techniques: In Vitro, SDS Page, Western Blot

Journal: Endocrinology

Article Title: Exercise induces a marked increase in plasma follistatin: evidence that follistatin is a contraction-induced hepatokine.

doi: 10.1210/en.2010-0868

Figure Lengend Snippet: FIG. 1. A, Plasma follistatin in relation to 3 h of bicycling exercise (n 5). The shaded area marks the 3 h period of exercise. B, Arterial and venous follistatin plasma concentrations during 2 h of one-legged knee-extensor exercise and the following recovery period (n 9). The shaded area marks the 2-h period of exercise. C, Follistatin mRNA expression in skeletal muscle. D, Follistatin mRNA expression in C2C12 muscle cells in response to EPS. E, Follistatin protein release from C2C12 muscle cells in response to EPS. *, P 0.05.

Article Snippet: The membranes were incubated overnight at 4 C with the primary antibody against follistatin (polyclonal goat antihuman follistatin, R&D Systems) (1:1000) in Tris-buffered saline with 0.1% Tween 20 and 5% skim milk, (Sigma-Aldrich, St. Louis, MO).

Techniques: Clinical Proteomics, Expressing

Journal: Endocrinology

Article Title: Exercise induces a marked increase in plasma follistatin: evidence that follistatin is a contraction-induced hepatokine.

doi: 10.1210/en.2010-0868

Figure Lengend Snippet: FIG. 2. A, Plasma follistatin in mice in relation to 1 h of swimming and the following recovery period (n 8 at each time point). B, Regulation of follistatin mRNA expression in mice in relation to 1 h of swimming and the following recovery period (n 8 at each time-point). The following tissues were analyzed: spleen, liver, subcutaneous (SubQ) and visceral fat (Vis), soleus, and gastrocnemius (Gastro) muscle, heart, and kidney. C, Western blot on liver tissue from mice performing 1 h of swimming exercise. Time points 1 h, 3 h, and 6 h are depicted as the relative proportion of the 0 h levels. *, P 0.05.

Article Snippet: The membranes were incubated overnight at 4 C with the primary antibody against follistatin (polyclonal goat antihuman follistatin, R&D Systems) (1:1000) in Tris-buffered saline with 0.1% Tween 20 and 5% skim milk, (Sigma-Aldrich, St. Louis, MO).

Techniques: Clinical Proteomics, Expressing, Western Blot

Journal: Endocrinology

Article Title: Exercise induces a marked increase in plasma follistatin: evidence that follistatin is a contraction-induced hepatokine.

doi: 10.1210/en.2010-0868

Figure Lengend Snippet: FIG. 4. A, Injection (ip) of 2 mg/kg epinephrine to mice (n 10 in each group). Plasma glucose before and after epinephrine injection. B, Injection (ip) of 2 mg/kg epinephrine to mice (n 10 in each group) Expression of follistatin in the liver in the control and epinephrine- stimulated groups. *, P 0.05. C, Hepatocytes treated with EPS- conditioned media from C2C12 myotubes. CON, Media from non-EPS- stimulated myotubes; MO, negative control consisting of media only; POS, TGF- were used as a positive control. Only TGF- could induce an increase in follistatin mRNA expression.

Article Snippet: The membranes were incubated overnight at 4 C with the primary antibody against follistatin (polyclonal goat antihuman follistatin, R&D Systems) (1:1000) in Tris-buffered saline with 0.1% Tween 20 and 5% skim milk, (Sigma-Aldrich, St. Louis, MO).

Techniques: Injection, Clinical Proteomics, Expressing, Control, Negative Control, Positive Control

Journal: Endocrinology

Article Title: Exercise induces a marked increase in plasma follistatin: evidence that follistatin is a contraction-induced hepatokine.

doi: 10.1210/en.2010-0868

Figure Lengend Snippet: FIG. 3. A, Infusion of recombinant IL-6 in healthy young men (n 7). A, Plasma IL-6 during a 3-h IL-6-infusion study. The shaded area marks the 3-h period of IL-6 infusion. B, Plasma follistatin during the 3-h IL-6- infusion study. No regulation of plasma follistatin. *, P 0.05. The shaded area marks the 3-h period of IL-6 infusion.

Article Snippet: The membranes were incubated overnight at 4 C with the primary antibody against follistatin (polyclonal goat antihuman follistatin, R&D Systems) (1:1000) in Tris-buffered saline with 0.1% Tween 20 and 5% skim milk, (Sigma-Aldrich, St. Louis, MO).

Techniques: Recombinant, Clinical Proteomics