Activin a

Activin A (Cat. No. 338-AC), GDF11 (Cat. No. 1958-GD), and BMP9 (Cat. No. 3209-BP) were purchased from R&D Systems. Assays were performed as previously described [14] (link). We constructed vectors pGL4.28 containing a Smad2/3-specific response element with Firefly luciferase as the reference gene and pGL4.26 containing a Smad1/5/8-response element with NanoLuc. The reporter construct was transfected into HEK293T cells using the Fugene HD reagent (Promega, Fitchburg, WI, USA). Transfected cells were seeded on Corning 384-well plates (15 μL containing 1 × 10⁴ cells in each well) in reaction medium, i.e., DMEM (Dulbecco's modified Eagle's medium) containing 0.1% BSA. After 2 h, 15 μL of reaction medium containing ligand (0.33 ng/mL of Activin A, or; 1 ng/mL of TGFβ, MSTN, or GDF11, or; 1.5 ng/mL of BMP9) and/or the indicated concentration of peptide was added to each well After additional incubation (4 h for Smad2/3 and 20 h for Smad1/5/8), cells were lysed to measure luciferase activity using the Bright-Glo Luciferase Assay System (Promega). Percent inhibition was calculated using the luminescence values from wells without peptide as 0% inhibition controls and values from wells without ligands as 100% inhibition controls. IC₅₀ values were calculated by Prism 5 (GraphPad Software, La Jolla, CA, USA).