ALK-1 Protein, Canine (HEK293, Fc)

ALK-1, also known as ACVRL1, is a type I receptor for TGF-β superfamily with 2 ligands, BMP9 and BMP10. ALK-1 is predominantly expressed in endothelial cells and plays a critical role in regulating developmental and pathological angiogenesis^[1][2]. ALK-1 Protein, Canine (HEK293, Fc) is produced in HEK293 cells with a C-Terminal Fc-tag. It consists of 119 amino acids (M1-K119).

ALK-1, also known as ACVRL1, is a type I receptor for TGF-β superfamily with 2 ligands, BMP9 and BMP10. ALK-1 is predominantly expressed in endothelial cells and plays a critical role in regulating angiogenesis^[1][2].
Mature human ALK-1 shares 89% amino acid sequence identity with mouse and rat ALK-1. While, mouse ALK-1 shares 96% aa sequence identity with rat ALK-1 protein.
ALK-1 is able to bind to TGF-β1 or activins in the presence of either TβR-II or activin type II receptors, respectively. However, ALK-1 does not elicit a specific transcriptional response. Thus, ALK-1 has been considered an “orphan” receptor. ALK-1 is a type I receptor that mediates signaling of BMP9 (bone morphogenetic protein) and BMP10, proteins in the TGF-β superfamily. Signaling through ALK-1 results in phosphorylation of the intracellular Smad 1/5/8 cascade which activates proangiogenic transcription factors such as ID1 and ID3. ALK-1 binds to TGF-β1 and phosphorylates Smad1 and Smad5. Overexpression of ALK-1 in HepG2 cells inhibits the ALK5-mediated TGF-β1 response. The balance between ALK-1 and ALK5 may be crucial for controlling the properties of endothelium during angiogenesis^[1]. BMP9/BMP10/ALK-1 signaling controlled the specific gene expression program and survival of Kupffer cells (KCs) through a Smad4-dependent pathway. Functionally, the loss of ALK-1 resulted in impaired capture of L. monocytogenes and overwhelming disseminated infections^[2].
ALK-1 is expressed in blood vessels during embryogenesis and adult stages. In addition, mutations of the ALK-1 gene have been linked to the type II hereditary hemorrhagic telangiectasia^[1]. ALK-1 inhibits BMP9-mediated Id-1 expression in human umbilical vein endothelial cells. In a chick chorioallantoic membrane assay, ALK-1 reduces VEGF-, FGF-, and BMP10-mediated vessel formation. In addition, ALK1 reduces tumor burden in mice receiving orthotopic grafts of MCF7 mammary adenocarcinoma cells^[3].

Recombinant human ALK-1 (5 μg/mL; preincubated for 3 minutes) abolishes Hey1, Hey2 and Id2 genes induction by serum in primary human endothelial cells^[4].

1.This product does not contain protein kinase domain.
2.Measured by its ability to inhibit BMP-9-induced alkaline phosphatase production by ATDC5 mouse chondrogenic cells. The ED₅₀ for this effect is 4.027 ng/mL in the presence of 2 ng/mL of rhBMP-9, corresponding to a specific activity is 2.483×10⁵ U/mg.

Materials
Cell Line for assay: ATDC5 cells
Culture Medium: DMEM
Phenylmethanesulfonyl fluoride (PMSF)
ALK-1 Protein, Canine (HEK293, Fc) (HY-P75516)
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 15,000 cells/well in a 24-well plate, with a total volume of 500 μL/well. Incubate the cells in a 37°C incubator with 5% CO₂ for 4 hours to allow cell attachment.
2. Remove the culture medium from the wells and add 500 μL of DMEM complete medium (containing 2 ng/mL BMP-9) with different concentrations of Canine ALK-1 protein to the 24-well plate, achieving final concentrations of Canine ALK-1 protein in each well of 0, 0.001, 0.01, 0.1, 0.5, 1, 2, 5, 10, 50, 100, 500, 2000 ng/mL.
3. Incubate the cells in a 37°C incubator with 5% CO₂ for 72 hours.
4. After the incubation period, lyse the cells using 200 μL of cell lysis buffer (containing a final concentration of 1 mM PMSF) . Allow the lysis buffer to act for 2 minutes, then collect the cell lysate, centrifμge, and take the supernatant.
5. Set up blank control wells, standard wells, and sample wells in a 96-well plate. The volumes of the standards used are 4, 8, 16, 24, 32, and 40 μL, respectively.
6. Gently pipette to mix and incubate at 37°C for 2 hours.
7. Add 100 μL of reaction stop solution to each well to terminate the reaction.
8. Measure the absorbance at 405 nm.
9. 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

E2R174 (G22-K119)

/

Partial

GNPVKPSRGPLLTCTCESPHCRGPTCQGTWCTVVLVREEGRHPQEHRGCGNLNEELCRGRPTEFVNHYCCYSPLCNHNVSLVLEATQIPPEQPKVDGQ

43-47 kDa

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

Lyophilized powder.

Lyophilized from a 0.22 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. For long term storage it is recommended to add a carrier protein (0.1% BSA, 5% HSA, 10% FBS or 5% Trehalose).

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]. S P Oh, et al. Activin receptor-like kinase 1 modulates transforming growth factor-beta 1 signaling in the regulation of angiogenesis. Proc Natl Acad Sci U S A. 2000 Mar 14;97(6):2626-31.  [Content Brief]

  [2]. Dianyuan Zhao, et al. ALK1 signaling is required for the homeostasis of Kupffer cells and prevention of bacterial infection. J Clin Invest. 2022 Feb 1;132(3):e150489.  [Content Brief]

  [3]. Dianne Mitchell, et al. ALK1-Fc inhibits multiple mediators of angiogenesis and suppresses tumor growth. Mol Cancer Ther. 2010 Feb;9(2):379-88.  [Content Brief]