ACVR2B Protein, Human/Cynomolgus (HEK293, hFc)

ACVR2B is a type II member of the TGF-β family of receptor Serine/Threonine kinases. ACVR2B binds to activins and growth differentiation factors (GDF), which in turn activate type I receptors, activating downstream molecule SMAD2/3^[1]. ACVR2B Protein, Human/Cynomolgus (HEK293, hFc) is produced in HEK293 cells with a C-Terminal hFc-tag.

Activin receptor type-2B (ACVR2B), also known as ActR-IIB and MGC116908, is an activin type II receptor. Type I receptors are essential for signaling; and type II receptors are required for binding ligands and for expression of type I receptors^[1].
The sequence of amino acids in ACVR2B proteins from different species is very stable, which leads to the conclusion that in the process of evolution, ACVR2B has been only slightly altered, and that both in humans and in animals, its function is similar.
Activins and growth differentiation factors (GDF) bind to ACVR2B, which in turn activate type I receptors such as activin receptor-like kinases (ALK) ALK4 and ALK5, activating downstream molecule SMAD2/3. SMADSs regulate a number of myogenic genes, such as myoD, myogenin, and Myf5, that are involved in cellular hypertrophy, proliferation, or differentiation. Noncanonical ACVR2B pathways have also been shown to regulate MAP kinases. ACVR2B blocks signaling of myostatin, its close homolog GDF11, as well as activin A, activin B, and BMP10^[1]. Activin A primarily binds to the type 1 receptors ALK4 or ALK7 in complex with ACVR2A or ACVR2B, causing activation of SMAD2 or SMAD3^[2].
In myeloma cells, BMP-6- and BMP-9-induced activation of SMAD1/5/8 through ACVR2A/ACVR2B/ALK2 is inhibited by activin A treatment^[2]. ACVR2B has been shown to preserve muscle mass and prolong survival in tumor hosts, and to increase bone mass in models of osteogenesis imperfecta and muscular dystrophy^[3].

Recombinant human ACVR2B (5 μg/mL) inhibits the effects of BMP-9 on INA-6 and IH-1 cells^[2].

1.This product does not contain protein kinase domain.
2.Measured by its ability to neutralize Activin-mediated inhibition on MPC11 cell proliferation. The ED₅₀ for this effect is 0.02-0.3496 µg/mL in the presence of 10 ng/mL recombinant Activin A.
3.Measured by its binding ability in a functional ELISA. Immobilized Inhibin Human, Mouse, Rat, Cynomolgus, Rhesus Inhibin beta A/Activin A at 2 μg/mL (100 μl/well) can bind Human ACVR2B hFc, the EC₅₀ of Human ACVR2B hFc is 12-60 ng/mL.

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  Measured by its ability to inhibit rhBMP-4-induced alkaline phosphatase production by ATDC5 mouse chondrogenic cells. The ED₅₀ for this effect is 0.03309 μg/mL in the presence of 15 ng/mL of Recombinant Human BMP‑4, corresponding to a specific activity is 3.022×10⁴ units/mg.

Materials
Cell Line for Assay: MPC-11 cells
Culture Conditions: Use DMEM high-glucose medium supplemented with 10% HS and 1% P/S. Cells are Cultured in a 37°C incubator with 5% CO₂
Penicillin-Streptomycin (100X) (HY-K1006)
Cell Counting Kit-8 (HY-K0301)
ACVR2B Protein, Human (HEK293, Fc) (HY-P72813)

Procedure
1. Seed cells at a density of 10,000 cells per well in a 96-well plate, with a total volume of 50 μL per well. (Do not seed cells in the outermost ring of the 96-well plate; instead, add an equal volume of PBS solution.)
2. Add 50 μL of DMEM complete medium (DMEM high-glucose medium + 10% HS + 1% P/S + 20 ng/mL Activin A) containing different concentrations of ACVR2B protein to the 96-well plate, achieving final concentrations of ACVR2B protein in each well of 0, 0.001, 0.01, 0.05, 0.1, 0.5, 1, 2.5, 5, 10, 25, 50, 100 μg/mL.
3. Incubate the cells in a 37°C incubator with 5% CO₂ for 72 hours.
4. After the incubation period, add 10 μL of CCK8 solution to each well under light-protected conditions and incubate in a 37°C incubator with 5% CO₂ for 2 hours.
5. Measure the OD value at 450 nm using a microplate reader.
6. Using GraphPad Prism software, plot a curve with OD values on the Y-axis and protein concentration on the X-axis to calculate the ED₅₀ value.

Human; Cynomolgus

HEK293

C-hFc

Q13705-1 (S19-T134)/XP_045242398.1 (S40-T155)

93  [NCBI]/102118668

Extracellular Domain

SGRGEAETRECIYYNANWELERTNQSGLERCEGEQDKRLHCYASWRNSSGTIELVKKGCWLDDFNCYDRQECVATEENPQVYFCCCEGNFCNERFTHLPEAGGPEVTYEPPPTAPT

Approximately 55-70 kDa due to the glycosylation.

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  ≥ 95%, as determined by reducing SDS-PAGE.

Lyophilized powder.

Lyophilized from a 0.2 μm filtered solution of PBS, pH 7.4.

<1 EU/μg, determined by LAL method.

It is not recommended to reconstitute to a concentration less than 100 μg/mL in ddH₂O.

Stored at -20°C for 2 years from date of receipt. After reconstitution, it is stable at 4°C for 1 week or -20°C for longer (with carrier protein). It is recommended to freeze aliquots at -20°C or -80°C for extended storage.

Room temperature in continental US; may vary elsewhere.

• [1]. Johanna Magga, et al. Systemic Blockade of ACVR2B Ligands Protects Myocardium from Acute Ischemia-Reperfusion Injury. Mol Ther. 2019 Mar 6;27(3):600-610.  [Content Brief]

  [2]. Oddrun Elise Olsen, et al. Activin A inhibits BMP-signaling by binding ACVR2A and ACVR2B. Cell Commun Signal. 2015 Jun 6;13:27.  [Content Brief]

  [3]. Rafael Barreto, et al. ACVR2B/Fc counteracts chemotherapy-induced loss of muscle and bone mass. Sci Rep. 2017 Oct 31;7(1):14470.  [Content Brief]