Journal: eLife

Article Title: Activin A forms a non-signaling complex with ACVR1 and type II Activin/BMP receptors via its finger 2 tip loop

doi: 10.7554/eLife.54582

Figure Lengend Snippet: ( A ) HEK293 cells harboring either a Smad2/3 or Smad1/5/8 reporter were treated with Activin A (1 nM), TGFβ1 (1 nM), or BMP6 (10 nM) in the presence of varying concentrations of SD208 or MAB222. ( top panel ) SD208 (TFGBR1 kinase inhibitor) inhibits Activin A-induced Smad2/3 signaling (IC 50 : 3.2 nM) and TGFβ1-induced Smad2/3 signaling (IC 50 : 1.4 nM) but does not affect BMP6-induced Smad1/5/8/signaling. ( bottom panel ) MAB222 (ACVR1B neutralizing antibody) inhibits Activin A induced Smad2/3 signaling (IC 50 : 37.4 nM) but leaves TGFβ1-induced Smad2/3 and BMP6-induced Smad1/5/8 signaling unaffected. ( B ) Smad-mediated signaling of HEK293 cells overexpressing ACVR1 was analyzed by immunoblotting. MAB222 plus SD208 inhibit Activin A-induced Smad2/3 phosphorylation but not BMP6-induced Smad1/5/8 phosphorylation. Consistent with prior observations, Activin A does not induce Smad1/5/8 phosphorylation via wild-type ACVR1. ( C ) Membrane-based sandwich immunoassay analysis of kinase phosphorylation (RnD Systems Proteome Profiler Human Phosphokinase Array Kit) was applied to the same cellular lysates utilized on panel B. ( D ) Quantitative analysis of Human Phospho-Kinase Array blots shown in panel C. The Activin A•ACVR1•type II receptor complex does not directly activate downstream signaling of the pathways included in this panel, as evidenced by the lack of increases in any of the phosphoproteins assayed therein. Figure 1—source data 1. Reporter assay data of HEK293 cells treated with MAB222 or SD208, and Phospho-Kinase array data of HEK293 cells treated with MAB222 + SD208 in the presence and absence of Activin A.

Article Snippet: In order to isolate signaling induced by Activin A only to the complex that it can form with ACVR1, HEK293 cells expressing ACVR1 were pretreated with 100 nM MAB222 (RnD Systems) plus 20 nM SD208 (BioVision) for 3 hr after overnight starvation.

Techniques: Western Blot, Phospho-proteomics, Membrane, Reporter Assay

Journal: eLife

Article Title: Activin A forms a non-signaling complex with ACVR1 and type II Activin/BMP receptors via its finger 2 tip loop

doi: 10.7554/eLife.54582

Figure Lengend Snippet: ( A ) Smad-mediated signaling of mouse embryonic stem cells (mES) cells where ACVR1 is not overexpressed was analyzed by immunoblotting using p-Smad1/5/8 and p-Smad2/3 antibodies. In order to isolate signaling induced by Activin A only to the complex that it can form with ACVR1, mES Acvr1b knockout (KO) cells were generated and used in comparison to the regular mES cells. mES cells were starved for 3 hr in the presence of 20 nM SD208. Then, signaling was induced with 10 nM Activin A or 10 nM BMP6 in the presence of 20 nM SD208 for 15 min. Both Acvr1b KO and SD208 inhibit Activin A-induced Smad2/3 phosphorylation but not BMP6-induced Smad1/5/8 phosphorylation. ( B ) Membrane-based sandwich immunoassay analysis of kinase phosphorylation (RnD Systems Proteome Profiler Human Phosphokinase Array Kit) was applied to the same cellular lysates utilized on panel A. ( D ) Quantitative analysis of Human Phospho-Kinase Array blots shown in panel B. The Activin A•ACVR1•type II receptor complex formed in mES Acvr1b KO cells didn’t not directly induce downstream phosphorylation of the kinases included in this panel indicating that the Activin A•ACVR1•type II receptor complex does not transduce signal.

Article Snippet: In order to isolate signaling induced by Activin A only to the complex that it can form with ACVR1, HEK293 cells expressing ACVR1 were pretreated with 100 nM MAB222 (RnD Systems) plus 20 nM SD208 (BioVision) for 3 hr after overnight starvation.

Techniques: Western Blot, Knock-Out, Generated, Comparison, Phospho-proteomics, Membrane

Journal: eLife

Article Title: Activin A forms a non-signaling complex with ACVR1 and type II Activin/BMP receptors via its finger 2 tip loop

doi: 10.7554/eLife.54582

Figure Lengend Snippet: ( A ) Activin A, from its structure with Follistatin 288 (2BOU) , was aligned into GDF11 structure from its ternary complex with TGFBR1 and ACVR2B (6MAC) . The ACVR1 model was aligned to TGFBR1 in the TGFBR1:GDF11:Acvr2B complex to give the energy minimized model. ( B ) Closer examination of the F2TL interaction in this model clearly shows Activin A F2TL residue D406 interacting electrostatically with both ACVR1 residues K78 and K82. ( C ) Substitution of Nodal F2TL into Activin A shows that F2TL coordination is disrupted.

Article Snippet: In order to isolate signaling induced by Activin A only to the complex that it can form with ACVR1, HEK293 cells expressing ACVR1 were pretreated with 100 nM MAB222 (RnD Systems) plus 20 nM SD208 (BioVision) for 3 hr after overnight starvation.

Techniques: Residue

Journal: eLife

Article Title: Activin A forms a non-signaling complex with ACVR1 and type II Activin/BMP receptors via its finger 2 tip loop

doi: 10.7554/eLife.54582

Figure Lengend Snippet: ( A ) Activin A F2TL muteins activate Smad2/3 signaling to similar levels as wild-type Activin A in HEK293 cells harboring a Smad2/3 reporter using firefly luciferase. ( B ) U20S cells expressing split beta-galactosidase fusions of corresponding type I and type II receptors were treated with a dose response of BMP7, Activin A, or Activin A.Nod.F2TL. Type I receptor binding was measured by luminescence in these receptor dimerization assays. Activin A.Nod.F2TL has reduced ability to dimerize ACVR1:ACVR2A receptors, while retaining wild type capacity to dimerize the ACVR1B:BMPR2 and TGFBR1:ACVR2B receptor pairs. The data presented are representative of at least three independent biological replicates. Three technical replicates were performed per experiment. Figure 4—source data 1. Reporter assay data of HEK293 cells and dimerization assay data of U20S cells treated with Activin A and Activin A F2TL muteins.

Article Snippet: In order to isolate signaling induced by Activin A only to the complex that it can form with ACVR1, HEK293 cells expressing ACVR1 were pretreated with 100 nM MAB222 (RnD Systems) plus 20 nM SD208 (BioVision) for 3 hr after overnight starvation.

Techniques: Luciferase, Expressing, Binding Assay, Reporter Assay

Journal: eLife

Article Title: Activin A forms a non-signaling complex with ACVR1 and type II Activin/BMP receptors via its finger 2 tip loop

doi: 10.7554/eLife.54582

Figure Lengend Snippet: Supernatants from CHO cells expressing Activin A with the pre or post-helix sequence from human Nodal were tested for activity in HEK293 Smad2/3 reporter cells. Both the Activin A.Nod.Pre and Activin A.Nod.Post supernatants display reduced activity compared to Activin A. The data presented are representative three independent biological and technical replicates.

Article Snippet: In order to isolate signaling induced by Activin A only to the complex that it can form with ACVR1, HEK293 cells expressing ACVR1 were pretreated with 100 nM MAB222 (RnD Systems) plus 20 nM SD208 (BioVision) for 3 hr after overnight starvation.

Techniques: Expressing, Sequencing, Activity Assay

Journal: eLife

Article Title: Activin A forms a non-signaling complex with ACVR1 and type II Activin/BMP receptors via its finger 2 tip loop

doi: 10.7554/eLife.54582

Figure Lengend Snippet: U20S cells expressing split beta-galactosidase fusions of corresponding type I and type II receptors were treated with a dose response of Activin A ligands. Type I receptor binding was measured by luminescence. ( A ) The finger two tip loop mutants have reduced ability to dimerize ACVR1 with ACVR2A, while retaining wild-type capacity to dimerize ( B ) ACVR1B with BMPR2 and ( C ) TGFBR1 with ACVR2B. The data presented are representative of three independent biological and technical replicates.

Article Snippet: In order to isolate signaling induced by Activin A only to the complex that it can form with ACVR1, HEK293 cells expressing ACVR1 were pretreated with 100 nM MAB222 (RnD Systems) plus 20 nM SD208 (BioVision) for 3 hr after overnight starvation.

Techniques: Expressing, Binding Assay

Journal: eLife

Article Title: Activin A forms a non-signaling complex with ACVR1 and type II Activin/BMP receptors via its finger 2 tip loop

doi: 10.7554/eLife.54582

Figure Lengend Snippet: For ( A ) and ( B ), 1000 RU of either ACVR1B-Fc or ACVR1-Fc were captured on a sensor chip. Ligands at 80 nM ( A ) or 1 μM ( B ) were injected, including Activin A, Activin B, Activin A.Nod.F2TL, BMP2, BMP4, BMP7, BMP9, BMP10, and BMP2/6 over ACVR1-Fc. ACVR1-Fc binds to BMP6, BMP7, and BMP9, but does not form a complex with Activin A, Activin B, Activin A.Nod.F2TL, BMP2, BMP4, BMP10, and BMP2/6. Ligands and corresponding binding curves are color matched. For ( C ) and ( D ), complexes of Ligand•Type II receptor extracellular domain were formed at 1:2.5 ratio and injected over ACVR1B-Fc and ACVR1-Fc. ( C ) Activin A•ACVR1B-Fc complex is detectable by SPR either alone or in the presence of monomeric extracellular domain of ACVR2A or ACVR2B. Activin A•ACVR2A and Activin A•ACVR2B, but not Activin A•BMPR2, complexes bound ACVR1-Fc at a stoichiometric ratio. ( D ) In contrast, none of the tested Activin A•Type II receptor complexes bound ACVR1-Fc. Ligand + / - Type II receptor and corresponding binding curves are color matched. The data shown here is a representative of two biochemical and two technical replicates.

Article Snippet: In order to isolate signaling induced by Activin A only to the complex that it can form with ACVR1, HEK293 cells expressing ACVR1 were pretreated with 100 nM MAB222 (RnD Systems) plus 20 nM SD208 (BioVision) for 3 hr after overnight starvation.

Techniques: Injection, Binding Assay

Journal: eLife

Article Title: Activin A forms a non-signaling complex with ACVR1 and type II Activin/BMP receptors via its finger 2 tip loop

doi: 10.7554/eLife.54582

Figure Lengend Snippet: ( A ) Activin A.Nod.F2TL is a less effective inhibitor of BMP7 signaling to Smad1/5/8 than wild-type Activin A. HEK293 cells harboring a Smad1/5/8 luciferase reporter construct were treated with varying concentrations of Activin A or Activin A.Nod.F2TL with a constant concentration of BMP7 (12 nM) to stimulate Smad1/5/8 signaling. Inhibition of BMP7 is reduced ~60 fold with Activin A.Nod.F2TL compared to Activin A. Using an Activin A antibody that blocks interaction with type II receptor (REGN2476), the remaining inhibition of BMP7 by Activin A.Nod.F2TL is lost. ( B ) Inhibition of type I receptor binding of Activin A with anti-Activin antibody H4H10442 shows a similar reduction in BMP inhibition to Activin A.Nod.F2TL. (The IC 50 s of Activin A and Activin A.Nod.F2TL are 1.4 × 10 −9 M and 9.7 × 10 −8 M, respectively. Insert in panel A shows a dose response of BMP7 on the HEK293 reporter cells, and the dotted lines represents the Smad1/5/8 signal induced by 12 nM BMP7 without inhibition by Activin A.). The data presented are representative of at least three independent biological replicates. Three technical replicates were performed per experiment. Figure 5—source data 1. Reporter assay data of HEK293 cells treated with anti-Activin A antibodies in the presence of BMP7+Activin A or BMP7+Activin A.Nod.F2TL.

Article Snippet: In order to isolate signaling induced by Activin A only to the complex that it can form with ACVR1, HEK293 cells expressing ACVR1 were pretreated with 100 nM MAB222 (RnD Systems) plus 20 nM SD208 (BioVision) for 3 hr after overnight starvation.

Techniques: Luciferase, Construct, Concentration Assay, Inhibition, Binding Assay, Reporter Assay

Journal: eLife

Article Title: Activin A forms a non-signaling complex with ACVR1 and type II Activin/BMP receptors via its finger 2 tip loop

doi: 10.7554/eLife.54582

Figure Lengend Snippet: ( A ) Schematic showing H4H10442 blocking Activin A (ActA) binding to type I receptor and REGN2476 preventing Activin A from binding to the type II receptor. ( B, C ) Varying concentrations of Activin A or Activin A.∆D406 were mixed with a constant concentration of BMP7 (12 nM) and applied to HEK293 cells stably transfected with the Smad1/5/8 reporter construct driving firefly luciferase. ( B ) Using REGN2476, we show the remaining inhibition of BMP7 by Activin A.∆D406 is lost when type II receptor binding is blocked. ( C ) Inhibition of type I receptor binding of wild-type Activin A shows further reduction in BMP inhibition compared to Activin A.∆D406 alone. Inhibition of BMP7 is reduced ~15 fold with Activin A.∆D406 compared to ~60 fold with the Activin A:H4H10442 complex. (The IC 50 s of Activin A and Activin A.∆D406 are 1.4 × 10 −9 M and 2.0 × 10 −8 M, respectively.) The data presented are representative of three independent biological and technical replicates.

Article Snippet: In order to isolate signaling induced by Activin A only to the complex that it can form with ACVR1, HEK293 cells expressing ACVR1 were pretreated with 100 nM MAB222 (RnD Systems) plus 20 nM SD208 (BioVision) for 3 hr after overnight starvation.

Techniques: Blocking Assay, Binding Assay, Concentration Assay, Stable Transfection, Transfection, Construct, Luciferase, Inhibition

Journal: eLife

Article Title: Activin A forms a non-signaling complex with ACVR1 and type II Activin/BMP receptors via its finger 2 tip loop

doi: 10.7554/eLife.54582

Figure Lengend Snippet: ( A ) Both H4H10442 and REGN2476 anti-Activin A antibodies block Activin A (10 nM) signaling through the Smad2/3 pathway in HEK293 CAGA-luciferase reporter cells. ( B ) In HEK293 Smad2/3 luciferase reporter cells, H4H10442 is a less effective inhibitor of the Activin A F2TL muteins (10 nM) when compared to wild-type Activin A. The data presented are representative of two independent biological and technical replicates. ( C ) Anti-Activin A monoclonal antibody H4H10442 appears to recognize an epitope that overlaps with the F2TL region of Activin A, as it does not bind as well to either Activin A.∆D406 or Activin A.Nod.F2TL. The ability of H4H10442 to bind the latter is particularly hampered. In contrast, REGN2476 bound similarly to all three ligands, consistent with the finding that the type II receptor-interacting regions of Activin A.∆D406 and Activin A.Nod.F2TL have not been altered. For dot blots, purified Activin A and Activin A muteins were serially diluted and applied to PVDF membranes using suction. Membranes were blocked using Odyssey blocking reagent and the hIGg4 was visualized using an IRDye 680RC conjugated goat anti-human secondary antibody (Li-cor). ( D ) Binding affinities and kinetic constants for binding purified anti-human Activin A monoclonal antibodies H4H10442 and REGN2476 to Activin A were determined using a real-time surface plasmon resonance biosensor at 37°C. Antibodies were captured on anti-human Fc sensor surfaces. Activin A-antibody association rates were measured by injecting 20 nM Activin A over the antibody captured surface. Both H4H10442 and REGN2476 bind Activin A with very high affinity, displaying KDs in low picomolar range.

Article Snippet: In order to isolate signaling induced by Activin A only to the complex that it can form with ACVR1, HEK293 cells expressing ACVR1 were pretreated with 100 nM MAB222 (RnD Systems) plus 20 nM SD208 (BioVision) for 3 hr after overnight starvation.

Techniques: Blocking Assay, Luciferase, Purification, Binding Assay, Bioprocessing, SPR Assay

Journal: eLife

Article Title: Activin A forms a non-signaling complex with ACVR1 and type II Activin/BMP receptors via its finger 2 tip loop

doi: 10.7554/eLife.54582

Figure Lengend Snippet: Varying concentrations of Follistatin and FSTL3 were preincubated with a constant concentration (10 nM) of Activin A or Activin A.Nod.F2TL ‘agonist only’ mutein. Activity of both Activin A and Activin A.Nod.F2TL were tested in HEK293 cells harboring the Smad2/3 luciferase reporter. Activity of both Activin A and Activin A.Nod.F2TL was blocked by both follistatin-288 ( A ) and follistatin-315 ( B ). ( C ) FSTL3 is a less effective inhibitor of Activin A.Nod.F2TL. The data presented are representative of at least three independent biological replicates. Three technical replicates were performed per experiment. Figure 6—source data 1. Reporter assay data of HEK293 cells treated with different isoforms of Follistatin in the presence of either Activin A or Activin A.Nod.F2TL.

Article Snippet: In order to isolate signaling induced by Activin A only to the complex that it can form with ACVR1, HEK293 cells expressing ACVR1 were pretreated with 100 nM MAB222 (RnD Systems) plus 20 nM SD208 (BioVision) for 3 hr after overnight starvation.

Techniques: Concentration Assay, Activity Assay, Luciferase, Reporter Assay

Journal: eLife

Article Title: Activin A forms a non-signaling complex with ACVR1 and type II Activin/BMP receptors via its finger 2 tip loop

doi: 10.7554/eLife.54582

Figure Lengend Snippet: Varying concentrations of Follistatin and FSTL3 were preincubated with a constant concentration (10 nM) of Activin A, Activin A.Nod.F2TL or Activin A. ∆.D406. Activity was tested in HEK293 cells harboring a Smad2/3 luciferase reporter. Activity of Activin A, ActivinA.Nod.F2TL and ActivinA. ΔD406 was blocked by both ( A ) Follistatin-288 and ( B ) Follistatin-315. ( C ) FSTL3 is a less effective inhibitor of ActivinA.Nod.F2TL and ActivinA.ΔD406 when compared to wild type Activin A. The data presented are representative of three independent biological and technical replicates.

Article Snippet: In order to isolate signaling induced by Activin A only to the complex that it can form with ACVR1, HEK293 cells expressing ACVR1 were pretreated with 100 nM MAB222 (RnD Systems) plus 20 nM SD208 (BioVision) for 3 hr after overnight starvation.

Techniques: Concentration Assay, Activity Assay, Luciferase

Journal: eLife

Article Title: Activin A forms a non-signaling complex with ACVR1 and type II Activin/BMP receptors via its finger 2 tip loop

doi: 10.7554/eLife.54582

Figure Lengend Snippet: HEK293 cells expressing FOP mutant ACVR1 (ACVR1[R206H]) were treated with a dose response of Activin A ligands. Both wild type Activin A and the Activin A F2TL muteins activate Smad1/5/8 pathway via ACVR1[R206H] receptor in the BRE-luciferase reporter assay. Notably, the Activin A.Nod.F2TL mutein is more active than either Activin A or Activin A.∆D406, consistent with the fact that Activin A.Nod.F2TL is not capable of forming a functional NSC with wild type ACVR1. The data presented are representative of three independent biological and technical replicates.

Article Snippet: In order to isolate signaling induced by Activin A only to the complex that it can form with ACVR1, HEK293 cells expressing ACVR1 were pretreated with 100 nM MAB222 (RnD Systems) plus 20 nM SD208 (BioVision) for 3 hr after overnight starvation.

Techniques: Expressing, Mutagenesis, Luciferase, Reporter Assay, Functional Assay

Journal: eLife

Article Title: Activin A forms a non-signaling complex with ACVR1 and type II Activin/BMP receptors via its finger 2 tip loop

doi: 10.7554/eLife.54582

Figure Lengend Snippet: ( A ) Representative µCT images of HO (yellow arrows) from tamoxifen-treated Acvr1 [R206H]FlEx/+ ; Gt(ROSA26)Sor CreERT2/+ mice taken 2 weeks after implantation of collagen sponges adsorbed with saline, 20 µg wild-type Activin A (ActA), or 20 µg Activin A.Nod.F2TL (ActA.Nod.F2TL). ( B ) Quantification of HO volume 2 weeks post-implantation. Saline, n = 2; ActA, n = 4; ActA.Nod.F2TL, n = 5. Each dot represents a single implantation with group mean (grey bar) and ± standard deviation (error bars) shown. Statistical significance was assessed by one-way ANOVA; **=p ≤ 0.01. ( C ) Representative µCT images of fore- and hindlimbs from SCID hosts 11 days post-transplantation of FOP FAPs (R206H-FAPs) in Geltrex alone, Geltrex contain 5 µg ActA, or Geltrex containing 5 µg ActA.Nod.F2TL. HO is pseudocolored beige for forelimbs and blue for hindlimbs. ( D ) Quantification of HO volume 11 days post-transplantation of R206H-FAPs. Geltrex only, n = 8; ActA, n = 14; n = 14; ActA.Nod.F2TL. Each dot represents a single transplantation with group mean (grey bar) and ± standard deviation (error bars) shown. Statistical significance was assessed by one-way ANOVA; ****=p ≤ 0.0001.

Article Snippet: In order to isolate signaling induced by Activin A only to the complex that it can form with ACVR1, HEK293 cells expressing ACVR1 were pretreated with 100 nM MAB222 (RnD Systems) plus 20 nM SD208 (BioVision) for 3 hr after overnight starvation.

Techniques: Saline, Standard Deviation, Transplantation Assay

Journal: eLife

Article Title: Activin A forms a non-signaling complex with ACVR1 and type II Activin/BMP receptors via its finger 2 tip loop

doi: 10.7554/eLife.54582

Figure Lengend Snippet: HEK293 cells expressing wild type ACVR1 were treated with 3 nM BMP6 containing a dose response of either ( A ) Activin B or ( B ) Activin AB. Activin antagonism of BMP6 signaling was measured by luminescence from a BRE-luciferase reporter. The data presented are representative of two independent biological and three technical replicates.

Article Snippet: In order to isolate signaling induced by Activin A only to the complex that it can form with ACVR1, HEK293 cells expressing ACVR1 were pretreated with 100 nM MAB222 (RnD Systems) plus 20 nM SD208 (BioVision) for 3 hr after overnight starvation.

Techniques: Expressing, Luciferase

Journal: Biology

Article Title: BMP-2-Driven Osteogenesis: A Comparative Analysis of Porcine BMSCs and ASCs and the Role of TGF-β and FGF Signaling.

doi: 10.3390/biology14060610

Figure Lengend Snippet: Figure 6. Receptor expressions of ALK2, ALK4, ALK6, ALK5, ALK7, BMPR-II in pASC. Grouped representation of the respective receptor expression in the course of osteogenic differentiation (OM +/−BMP-2). From day 19, there is a significant induction of ALK 2, ALK 6, and ALK 5 with the addition of BMP-2. BMPR-II expression in the OM group decreased in OM and tended to stay increased under BMP-2 supplementation from day 19, but was not considered significant (* p ≤0.05, ** p ≤0.01; n = 6, BMP-2 450 ng/mL).

Article Snippet: The respective conjugated antibodies were used for the expressions of ALK3 (Cat. No.: AF436), ALK 5 (Cat. No.: FAB5871), ALK6 (Cat. No.: FAB5051A), TGF-β2-RII (Cat. No.: FAB532P), ALK7 (Cat. No.: FAB77491A), ALK2 (Cat. No.: AF637), ALK4 (Cat. No.: MAB2221), and BMPR-II (Cat. No.: AF811) (by R&D Systems, Minneapolis, MN, USA), and the pASCs and pBMSCs were compared for their expressions of the specific surface antigens CD45 (Cat. No.: MCA1568GA, BioRad, Hercules, CA, USA), HLA-DR (human leukocyte antigen–antigen D-related surface molecule) (Cat. No.: MCA2314F, Bio-Rad, Hercules, CA, USA), CD29 (Cat. No.: 561,496, BD Pharmingen, Franklin Lakes, NJ, USA), CD79alpha (Bio-Rad, Cat. No.: MCA2538GA), CD14 (Cat. No.: MCA1568GA, Bio-Rad, Hercules, CA, USA), CD31 (Cat. No.: AF3387, R&D Systems, Minneapolis, MN, USA), CD105 (Cat. No.: NB110-58718APC, Novus Biologicals, Minneapolis, MN, USA), CD26 (, Cat. No.: NB600-552APC, Novus Biologicals, Minneapolis, MN, USA), CD73 (, Cat. No.: AF4488, R&D Systems, Minneapolis, MN, USA), CD90 (Cat. No.: 559,869, BD Pharmingen, Franklin Lakes, NJ, USA), CD34 (Cat. No.: 81289, abcam, Cambridge, UK), and CD44 (Cat. No.: 5531, BD Pharmingen, Franklin Lakes, NJ, USA).

Techniques: Expressing

Journal: Anticancer research

Article Title: Potential roles of activin in head and neck squamous cell carcinoma progression in epithelial-mesenchymal transition, metastasis, and mortality

doi: 10.21873/anticanres.16733

Figure Lengend Snippet: The canonical activin A pathway. Activin A is composed of inhibin βA subunits (βA) and binds to activin receptor type II and IIB (ACVR2/2B). Inhibin, composed of a βA and α subunit, competitively binds and sequesters ACVR2/2B, ultimately inhibiting the activin axis. Contrariwise, activin binding ultimately forms a Smad transcriptions complex (comprised of Smad 2, 3 and 4), the phosphorylation of activin receptor type IB (ACVR1B) subsequently stimulating and activating the Smad transcription complex, thereby eliciting downstream cellular behaviors such as proliferation inhibition, apoptosis, and epithelial mesenchymal transition. Of note, activin may have proliferative effects outside of this axis, as described in the introduction and discussion.

Article Snippet: Antibody Clone Source Dilution ACVR1B (MAB222) Monoclonal R & D Systems 1:200 ACVR2 (AF340) Polyclonal R & D Systems 1:100 ACVR2B (AF339) Polyclonal R & D Systems 1:80 INHA (MCA951S) Monoclonal Biorad 1:800 INHBA (Serotec/Biorad) Monoclonal Biorad 1:100 INHBB (Serotec/Biorad) Monoclonal Biorad 1:100 Open in a separate window Immunohistochemistry MTT assay Cell proliferation was determined by MTT incorporation.

Techniques: Binding Assay, Phospho-proteomics, Inhibition

Journal: Anticancer research

Article Title: Potential roles of activin in head and neck squamous cell carcinoma progression in epithelial-mesenchymal transition, metastasis, and mortality

doi: 10.21873/anticanres.16733

Figure Lengend Snippet: Immunohistochemistry expression of inhibin subunits (INHA, INHBA, INHBB) and activin receptors (ACVR1B, ACVR2, ACVR2B) in five normal, 15 oral premalignant (OPL) and 12 HNSCC tumor tissue samples. Chi-square tests were employed for analysis with p <0.05 being significant; diffuse and focal positivity were scored as positive. Premalignant and malignant lesions demonstrated a statistically significant increase in the prevalence of ligand inhibin βA (INHBA) (χ 2 (2, N = 32) = 18.98, p < .0001) (Row 6) as well as ACVR1B (χ 2 (2, N = 32) = 11.52, p < .0032) (Row 11). There was also a decreased prevalence of ACVR2B among pre-malignant and malignant lesions in comparison to normal mucosa (χ 2 (2, N = 32) = 0.0018, p < .0018) (Row 13).

Article Snippet: Antibody Clone Source Dilution ACVR1B (MAB222) Monoclonal R & D Systems 1:200 ACVR2 (AF340) Polyclonal R & D Systems 1:100 ACVR2B (AF339) Polyclonal R & D Systems 1:80 INHA (MCA951S) Monoclonal Biorad 1:800 INHBA (Serotec/Biorad) Monoclonal Biorad 1:100 INHBB (Serotec/Biorad) Monoclonal Biorad 1:100 Open in a separate window Immunohistochemistry MTT assay Cell proliferation was determined by MTT incorporation.

Techniques: Immunohistochemistry, Expressing, Comparison

Journal: Anticancer research

Article Title: Potential roles of activin in head and neck squamous cell carcinoma progression in epithelial-mesenchymal transition, metastasis, and mortality

doi: 10.21873/anticanres.16733

Figure Lengend Snippet: Immunohistochemistry

Article Snippet: Antibody Clone Source Dilution ACVR1B (MAB222) Monoclonal R & D Systems 1:200 ACVR2 (AF340) Polyclonal R & D Systems 1:100 ACVR2B (AF339) Polyclonal R & D Systems 1:80 INHA (MCA951S) Monoclonal Biorad 1:800 INHBA (Serotec/Biorad) Monoclonal Biorad 1:100 INHBB (Serotec/Biorad) Monoclonal Biorad 1:100 Open in a separate window Immunohistochemistry MTT assay Cell proliferation was determined by MTT incorporation.

Techniques:

Journal: Nature Communications

Article Title: Loss of ALK4 promotes cancer progression through regulating TGF-β receptor N-glycosylation

doi: 10.1038/s41467-025-67563-1

Figure Lengend Snippet: a MDA-MB-231 cells stably expressing a control shRNA (shNTC), shRNA targeting ALK4 (shALK4), or shRNA-resistant ALK4 (rALK4) were injected into the tail vein of nude mice ( n = 10 per group). Pulmonary lesions were detected by bioluminescent imaging. Total bioluminescence at the end of the experiment (week 7) is presented with representative images. Statistical analysis was performed using nonparametric one-way ANOVA (Kruskal–Wallis test) followed by Dunn’s multiple comparisons test. Data are presented as mean values ± SEM. b–f LM2 cells expressing pBABE or pBABE-ALK4 vectors were injected into the tail vein of nude mice ( n = 15 per group). Pulmonary lesions were imaged by bioluminescent imaging at the indicated times. b Representative images of LM2 pBABE- and pBABE-ALK4-injected mice. c Normalized bioluminescence of pulmonary lesions at the indicated times. Data are presented as mean values ± SD. Data were analyzed by ordinary two-way ANOVA with Tukey’s multiple comparisons test. d Average number of palpable pulmonary lesions with representative images of the lungs. Data are presented as mean values ± SEM. e Average lung weight at week 7 of LM2 pBABE- and pBABE-ALK4-injected mice. Data are presented as mean values ± SEM. f Kaplan–Meier survival curves for LM2 pBABE- and pBABE-ALK4-injected mice. g–k HPNE pancreatic cells stably expressing shNTC or shALK4 were orthotopically injected into the tail of mouse pancreata ( n = 8 per group). g Total bioluminescence and representative images of the mice 7 weeks after injection. h Primary pancreatic tumors from mice injected with HPNE cells expressing shNTC or shALK4 were dissected and weighed. i H&E staining of primary pancreatic tumors from mice bearing HPNE cells expressing shNTC or shALK4.Scale bar = 150 μm. j Representative images of spontaneous metastases observed in mice bearing orthotopic HPNE pancreatic cells expressing shALK4. Scale bar = 50 μm. k Quantification of spontaneous metastases in mice bearing orthotopic HPNE cells expressing shNTC or shALK4, data were analyzed by a two-sided Chi-square test. Other data comparing two groups were analyzed using a nonparametric two-tailed Mann–Whitney test.

Article Snippet: The ALK4 WT (Cat# 80879), ALK4 CA (Cat# 27223), TβRII DN (Cat# 12640), TβRI (Cat# 14831), and TβRII (Cat# 11766) DNA plasmids were purchased from Addgene.

Techniques: Stable Transfection, Expressing, Control, shRNA, Injection, Imaging, Staining, Two Tailed Test, MANN-WHITNEY

Journal: Nature Communications

Article Title: Loss of ALK4 promotes cancer progression through regulating TGF-β receptor N-glycosylation

doi: 10.1038/s41467-025-67563-1

Figure Lengend Snippet: a MDA-MB-231 and b PANC-1 CRISPR non-targeting control (NTC) cells and two CRISPR ALK4 KO clones for each cell line were analyzed for expression of EMT markers and β-actin by Western blotting. One representative out of three independent replicates is shown. c Soft agar colony formation assay of PANC-1 NTC cells and two ALK4 KOs clones ( n = 4 independent experiments), scale bar = 200 nm. Data were analyzed using ordinary one-way ANOVA followed by Dunnett’s multiple comparisons test. Cell migration was assessed by transwell migration assay in PANC-1 ( d ) and HPNE ( e ) cells expressing shNTC or shALK4 ( n = 3 biological replicates). f MDA-MB-231 cells expressing shNTC, shALK4, or shALK4 and shRNA-resistant ALK4 ( n = 5 biological replicates) were analyzed using transwell migration assays. Cell invasion was assessed using Matrigel transwell assays in PANC-1 ( n = 10 biological replicates) ( g ) and MDA-MB-231( n = 4 biological replicates) ( h ) cells expressing shNTC or shALK4. LM2 cells expressing pBABE or pBABE-ALK4 ( n = 3 biological replicates) were analyzed for migration ( i ) and invasion ( j ). k MDA-MB-231 cells expressing shNTC or shALK4 were sparsely seeded and subjected to live cell imaging to track migration. Data from 23 cells for each line are shown, with directional persistence calculated in ( l ). Pancreatic (PAAD) and breast (BRCA) cancer patient data from TCGA, CPTAC-PAAD, QCMG-PAAD, and METABRIC-BRCA were stratified based on ALK4 expression. Patients in the bottom quartile (low ALK4) and top quartile (high ALK4) of ALK4 expression from these cohorts were selected. Gene expression data were used for gene set enrichment analysis (GSEA) with cancer-associated gene signatures related to EMT ( m ) or invasive cancer phenotypes ( n ) that were significantly enriched in the ALK4-low group. Normalized enrichment scores (NES) with p -values < 0.05 are indicated by the colored symbols, as defined in the plot legends. The soft agar, migration, and invasion assays were quantified in a blinded manner. Data comparing two groups were analyzed using two-tailed Student’s t -tests. Data are presented as mean values ± SEM.

Article Snippet: The ALK4 WT (Cat# 80879), ALK4 CA (Cat# 27223), TβRII DN (Cat# 12640), TβRI (Cat# 14831), and TβRII (Cat# 11766) DNA plasmids were purchased from Addgene.

Techniques: CRISPR, Control, Clone Assay, Expressing, Western Blot, Soft Agar Assay, Migration, Transwell Migration Assay, shRNA, Live Cell Imaging, Gene Expression, Two Tailed Test

Journal: Nature Communications

Article Title: Loss of ALK4 promotes cancer progression through regulating TGF-β receptor N-glycosylation

doi: 10.1038/s41467-025-67563-1

Figure Lengend Snippet: HPNE ( a ) and PANC-1 ( b ) cells expressing shNTC or shALK4 were serum-starved for 3 h and then treated with vehicle, 100 pM activin A, 100 pM TGF-β1, 100 ng/ml Nodal, 1 nM GDF5, or 5% serum for 30 min. Protein lysates were analyzed by Western blotting. One representative out of three independent replicates is shown. c MDA-MB-231 NTC cells and two isogenic ALK4 KO lines were serum-starved for 3 h and then treated with vehicle or 100 pM TGF-β1 for 30 min. Protein lysates were analyzed by Western blotting. One representative out of three independent replicates is shown. d Nuclear localization of pSmad2 in NTC-TGF-β1 ( n = 165 cells), NTC + TGF-β1 ( n = 324 cells), ALK4 KO-TGF-β1 ( n = 93 cells), and ALK4 KO + TGF-β1 ( n = 194 cells) groups was assessed by immunofluorescent microscopy. Scale bar = 25 µm. Nuclear blob counts were quantified using BlobFinder v3.2. The central line marks the median, the box extends from the 25th to the 75th percentiles. The whiskers go from min to max. e Pancreatic (PAAD) and breast (BRCA) cancer patients from the TCGA, CPTAC-PAAD, QCMG-PAAD, and METABRIC-BRCA cohorts were stratified into bottom quartile (low ALK4) and top quartile (high ALK4) groups. Gene set enrichment analysis (GSEA) of TGF-β pathway-related gene signatures was performed, with normalized enrichment scores (NES, p < 0.05) indicated by the colored symbols, as defined in the plot legend. f Five out of seven TGF-β target genes are significantly negatively correlated with ACVR1B in the TCGA breast cancer expression dataset ( n = 960). g Seven TGF-β target genes are significantly and negatively correlated with ACVR1B in the TCGA pancreatic cancer expression dataset ( n = 149). h–j MDA-MB-231 CRISPR NTC and ALK4 KO cells, with or without dominant-negative TβRII (DN-TβRII), were serum-starved for 3 h and treated with vehicle or 100 pM TGF-β1 for 30 min. h Protein lysates were analyzed by Western blotting. i SERPINE1 expression was measured by qRT-PCR ( n = 3 biological replicates). j Cell migration was assessed using transwell migration assays ( n = 4 biological replicates). For experiments with two independent variables, statistical analyses were performed using ordinary two-way ANOVA with Tukey’s multiple comparisons test. Data are presented as mean values ± SEM.

Article Snippet: The ALK4 WT (Cat# 80879), ALK4 CA (Cat# 27223), TβRII DN (Cat# 12640), TβRI (Cat# 14831), and TβRII (Cat# 11766) DNA plasmids were purchased from Addgene.

Techniques: Expressing, Western Blot, Microscopy, CRISPR, Dominant Negative Mutation, Quantitative RT-PCR, Migration

Journal: Nature Communications

Article Title: Loss of ALK4 promotes cancer progression through regulating TGF-β receptor N-glycosylation

doi: 10.1038/s41467-025-67563-1

Figure Lengend Snippet: a–c Cell surface levels of TGF-β receptors were assessed using I 125 -TGF-β binding and crosslinking assays. MDA-MB-231 ( a ) and PANC-1 ( b ) cells expressing shNTC control or shALK4 were serum-starved for 3 h and treated with 100 pM TGF-β1 for the indicated times. c 293T cells were transfected with expression plasmids for TβRII and/or TβRI, along with wild-type (WT), constitutively active (CA), or dominant-negative (DN) ALK4. ALK4 and β-actin were analyzed. d LM2 cells were transfected with expression plasmids for TβRII, along with wild-type (WT) or dominant-negative (DN) ALK4. Biotinylated cell surface proteins were analyzed for TβRII, and whole cell lysates were analyzed for ALK4 and β-actin using Western blotting. e LM2 cells expressing pBABE control or pBABE-ALK4 were serum-starved for 3 h and treated with 100 pM TGF-β1 for the indicated times. Cell surface levels of TGF-β receptors were assessed using I 125 -TGF-β binding and crosslinking assays. f 293T cells were transfected with WT ALK4 as indicated, and total cell lysates were processed with PNGase F and assessed by Western blotting. g PANC-1 and MDA-MB-231 cells expressing shNTC or shALK4 were lysed. Total cell lysates were processed with PNGase F or EndoH and assessed by Western blotting. h MDA-MB-231 cells stably expressing shNTC or shALK4 were treated with tunicamycin at the indicated concentrations. Cell surface levels of TGF-β receptors were assessed using an I 125 -TGF-β binding and crosslinking assay (top). Total TβRII and β-actin were assessed by Western blotting. i MDA-MB-231 cells expressing shNTC or shALK4 were treated with 10 nM cycloheximide (CHX) for the indicated times to block synthesis of new proteins. Cells were incubated with 50 ng/ml tunicamycin for 4 h prior to harvesting. Whole-cell lysates were assessed for TβRII and β-actin by Western blotting. Quantification was performed using ImageJ. For in vitro analysis, each experiment was done with at least three independent biological replicates.

Article Snippet: The ALK4 WT (Cat# 80879), ALK4 CA (Cat# 27223), TβRII DN (Cat# 12640), TβRI (Cat# 14831), and TβRII (Cat# 11766) DNA plasmids were purchased from Addgene.

Techniques: Binding Assay, Expressing, Control, Transfection, Dominant Negative Mutation, Western Blot, Stable Transfection, Blocking Assay, Incubation, In Vitro

Journal: Nature Communications

Article Title: Loss of ALK4 promotes cancer progression through regulating TGF-β receptor N-glycosylation

doi: 10.1038/s41467-025-67563-1

Figure Lengend Snippet: a Quantitative proteomic analysis of whole-cell lysates from PANC-1 control (NTC) and two ALK4 CRISPR knockout (ALK4 KO) lines. Heatmap showing the expression of differentially expressed proteins (DEPs, p < 0.05) between ALK4 KO and control samples. Colors represent relative expression values in log fold change (logFC). b Functional annotation and pathway enrichment analysis of the top 125 upregulated DEPs in ALK4 KO samples compared to controls using DAVID Bioinformatics. Significant gene sets are shown with circle size indicating the number of genes and color indicating the transformed p-value. Fold enrichment is represented on the x -axis. c–f Gene set enrichment analysis (GSEA) plots for DEPs (KO vs. WT) showing significant enrichment in indicated gene signatures. NES: normalized enrichment score. g Protein–protein interaction network of the top-regulated proteins in ALK4 KO cells, generated using STRING analysis. Interactions with a score > 0.5 are depicted, with line color indicating the type of interaction (cyan-curated databases; pink-experimentally determined; blue-gene co-occurrence; khaki-text mining; black-co-expression; light blue-protein homology). Dots represent the pathways/biology as indicated.

Article Snippet: The ALK4 WT (Cat# 80879), ALK4 CA (Cat# 27223), TβRII DN (Cat# 12640), TβRI (Cat# 14831), and TβRII (Cat# 11766) DNA plasmids were purchased from Addgene.

Techniques: Control, CRISPR, Knock-Out, Expressing, Functional Assay, Transformation Assay, Generated

Journal: Nature Communications

Article Title: Loss of ALK4 promotes cancer progression through regulating TGF-β receptor N-glycosylation

doi: 10.1038/s41467-025-67563-1

Figure Lengend Snippet: a Western blot (left) and qRT-PCR (right) analysis of protein and LGALS3 mRNA expression in PANC-1 NTC and ALK4 KO cells ( n = 4 per group). b Flow cytometric analysis of MGAT5-modified glycans labeled with PHA-L lectin in PANC-1 NTC ( n = 14), ALK4 KO1 ( n = 12), ALK4 KO2 ( N = 14), and ALK4 KO3 ( n = 13) cells. c qRT-PCR analysis of MGAT5 expression in PANC-1 NTC and ALK4 KO cells ( n = 4 per group). d Magnetic beads were coated with PHA-L lectin and incubated with whole-cell lysates from PANC-1 NTC or ALK4 KO cells. Eluted samples were analyzed for TβRII. Whole-cell lysates (WCL) were analyzed for TβRII and β-actin. e PANC-1 control and ALK4 KO cells transfected with control or MGAT5-specific siRNA. After 2 days, cells were transfected again for an additional 3 days, serum-starved for 3 h, and treated with 100 pM TGF-β1 for 30 min before harvesting. Protein lysates were assessed for expression of the indicated proteins using western blotting. f Western blot analysis of protein expression in PANC-1 NTC and ALK4 KO cells transfected with control or galectin-3-specific siRNA for 5 days. Cells were treated with 100 pM TGF-β1 for 30 min before harvesting. g Internalization of TβRII in PANC-1 NTC ( n = 3) and ALK4 KO cells ( n = 5). Quantification of internalized TβRII under the indicated condition is shown at the bottom. h Resullts of soft agar colony formation assay of indicated groups ( n = 6 per group), scale bar = 200 nm. i–k In vivo pulmonary lesions in NSG mice injected via tail vein with PANC-1 NTC, PANC-1 NTC crMGAT5 KO, ALK4 KO, or ALK4 KO crMGAT5 KO cells ( n = 10 per group). Pulmonary lesions were imaged via bioluminescent imaging in a blinded manner ( i and j ). k Bioluminescent imaging of lungs from the top three mice (highest bioluminescence) from each group at week 7. For in vitro analysis, each experiment was done with at least 3 independent biological replicates. For multiple comparisons with one independent variable, ordinary one-way ANOVA was used, followed by Dunnett’s multiple comparisons test. For experiments with two independent variables, ordinary 2-way ANOVA was used, followed by Tukey’s multiple comparisons test. Data are presented as mean values ± SEM.

Article Snippet: The ALK4 WT (Cat# 80879), ALK4 CA (Cat# 27223), TβRII DN (Cat# 12640), TβRI (Cat# 14831), and TβRII (Cat# 11766) DNA plasmids were purchased from Addgene.

Techniques: Western Blot, Quantitative RT-PCR, Expressing, Modification, Labeling, Magnetic Beads, Incubation, Control, Transfection, Soft Agar Assay, In Vivo, Injection, Imaging, In Vitro

Journal: Nature Communications

Article Title: Loss of ALK4 promotes cancer progression through regulating TGF-β receptor N-glycosylation

doi: 10.1038/s41467-025-67563-1

Figure Lengend Snippet: Dot plots of the correlation between ACVR1B and ETS1 expression (left) and between MGAT5 and ETS1 expression (right) using the TCGA pancreatic ( a ) and breast ( b ) cancer databases. c Spearman correlation coefficients between the indicated genes in the TCGA pancreatic and breast cancer databases. NS not significant. d qRT-PCR analysis of ETS1 mRNA levels in PANC-1 NTC and ALK4 KO cells. qRT-PCR analysis of ETS1 ( e ) and MGAT5 ( f ) PANC-1 NTC and ALK4 KO cells transfected with control or ETS1-specific siRNA for 48 h. Results were repeated with three independent biological replicates. For experiments with two independent variables, data were analyzed using ordinary two-way ANOVA followed by Tukey’s multiple comparisons test. For comparison between two groups, data were analyzed using two-tailed Student’s t tests. Data are presented as mean values ± SEM.

Article Snippet: The ALK4 WT (Cat# 80879), ALK4 CA (Cat# 27223), TβRII DN (Cat# 12640), TβRI (Cat# 14831), and TβRII (Cat# 11766) DNA plasmids were purchased from Addgene.

Techniques: Expressing, Quantitative RT-PCR, Transfection, Control, Comparison, 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: (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