BMPR1A/ALK-3 Protein, Canine (HEK293, Fc)

BMPR1A/ALK-3 protein (ACVRLK3) is the receptor bone morphogenetic protein (BMP) type I receptors, widely expressed in tissues^[1]. BMPR1A/ALK-3 protein mediates iron metabolism factor hepcidin expression, interacts with GDF5/6 to regulate chondrocyte differentiation and adipogenesis^[2][3]. BMPR1A-ID2/ZEB1-TGFBR2 signaling axis could serve as a potentia target for pulmonary arterial hypertension (PAH) and other endothelial-mesenchymal transition (EndoMT)-related vascular disorders^[4]. BMPR1A/ALK-3 Protein, Canine (HEK293, Fc) is a recombinant protein produced in the HEK293 cells with C-terminal hFc-tag.

ALK-3 (BMPR1A; ACVRLK3) is the receptor bone morphogenetic protein (BMP) type I receptors, for BMP2, BMP4, GDF5 and GDF6. Among BMP type I receptors, ALK-2 and 3 are widely expressedin tissues, while ALK-1 is more selectively expressed in endothelial cells (ECs)^[1]. Hepcidin, the main regulator of iron metabolism, is synthesized and released by hepatocytes in response to increased body iron concentration and inflammation. BMP/ALK/SMAD pathway controls hepcidin expression, while BMP type I receptors ALK-2 and ALK-3 are responsible for iron-dependent hepcidin upregulation and basal hepcidin expression, respectively, to avoid low hepcidin which causes iron overload or high hepcidin levels which induce iron-restricted erythropoiesis^[2]. ALK-3 positively regulates chondrocyte differentiation through GDF5 interaction and mediates induction of adipogenesis by GDF6^[3]. ALK-3 protein shows function for the initiation of chondrogenesis, for regulating differentiation along the chondrogenic lineage, and for endochondral bone formation^[5]. Components of BMP signaling have been implicated in both pathogenesis of pulmonary arterial hypertension (PAH) and endothelial-mesenchymal transition (EndoMT), and BMPR1A is key to maintain endothelial identity and to prevent excessive EndoMT. BMPR1A-ID2/ZEB1-TGFBR2 signaling axis could serve as a potential novel target for PAH and other EndoMT-related vascular disorders^[4].

1.This product does not contain protein kinase domain.
2.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.

• 
  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 used for assay: MPC-11
Cell culture medium: DMEM +10% HS+1% P/S (DMEM complete medium)
BMP-4 (HY-P7007)
BMPR1A/ALK-3 Protein
Phenylmethanesulfonyl fluoride (PMSF)
Penicillin-Streptomycin (100×), Sterile (HY-K1006)
Cell Counting Kit-8 (CCK8 solution, HY-K0301)
Alkaline Phosphatase Assay Kit: Contains assay buffer, chromogenic substrate, p-nitrophenol solution and reaction stop solution

Procedure
1. Seed cells at a density of 3000 cells/well in a 96-well plate, with a total volume of 100 μL/well. (Do not seed cells in the outermost ring of the 96-well plate; instead, add an equal volume of PBS solution.)
2. Prepare different concentrations of Canine ALK-3 protein in complete DMEM medium (containing BMP-4 at a final concentration of 15 ng/mL), yielding final Canine ALK-3 concentrations of 0, 0.00001, 0.0001, 0.001, 0.01, 0.025, 0.05, 0.1, 0.25, 0.5, 1, 10, 25, 50 μg/mL. Incubate at 37°C in a 5% CO₂ incubator for 72 hours.
3. At the end of the incubation period, cells were lysed using 200 μL of cell lysis buffer (containing 1 mM PMSF). After incubation for 2 minutes, the cell lysate was collected, centrifuged, and the supernatant was retrieved.
4. Set up blank control wells, standard wells, and sample wells in a 96-well plate. The configuration for each group is as follows, with standard volume x being 4, 8, 16, 24, 32, and 40 μL respectively.
Blank control wells: 50 μL assay buffer, 50 μL chromogenic substrate;
Standard wells: x μL standard working solution, (100-x) μL detection buffer;
Sample wells: y μL sample, (50-y) μL detection buffer, 50 μL colour developing substrate.
5. Gently mix by pipetting up and down, then incubate at 37°C for 30 minutes.
6. Add 100 μL reaction stop solution to each well to terminate the reaction.
7. Measure absorbance at 405 nm.
8. 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.

Canine

HEK293

C-hFc

A0A2U3WMM9/NP_001138622.1 (Q24-R152)

489077  [NCBI]

Partial

QNLDSMLHGTGMKSDSDQKKSENGVTLAPEDTLPFLKCYCSGHCPDDAINNTCITNGHCFAIIEEDDQGETTLASGCMKYEGSDFQCKDSPKAQLRRTIECCRTNLCNQYLQPTLPPVVIGPFFDGSIR

Approximately 55 kDa.

• 
  ≥ 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]. Yang P, et al. The role of bone morphogenetic protein signaling in vascular calcification. Bone. 2020 Dec;141:115542.  [Content Brief]

  [2]. Traeger L, et al. HFE and ALK3 act in the same signaling pathway. Free Radic Biol Med. 2020 Nov 20;160:501-505.  [Content Brief]

  [3]. Miyazawa K, et al. Regulation of TGF-β Family Signaling by Inhibitory Smads. Cold Spring Harb Perspect Biol. 2017 Mar 1;9(3):a022095.  [Content Brief]

  [4]. Lee HW, et al. BMPR1A Promotes ID2-ZEB1 Interaction to Suppress Excessive Endothelial to Mesenchymal Transition. Cardiovasc Res. 2022 Sep 27:cvac159.  [Content Brief]

  [5]. Jing J, et al. Bmpr1a Signaling in Cartilage Development and Endochondral Bone Formation. Vitam Horm. 2015;99:273-91.  [Content Brief]