Fig 1: Antagonist activities of peptides in Smad reporter assays. (A) Receptor activation by TGFβ, activin A, MSTN, or GDF11 was detected by Smad2/3 reporter assays. Receptor activation by BMP9 was detected by Smad1/5/8 reporter assays. Percent inhibition was calculated as described in the “Materials and methods” section. (B) Gene expression analysis of activin receptors in the HEK293T cells that were used in the Smad reporter assays. Target mRNA expressions were normalized to the expression of the internal control gene 36B4. (C) Ligand-binding activities of ActRIIB-Fc and ActRIIA-Fc as determined by plate ELISA.
Fig 2: ActE signals via the activin type II receptors.(A) Schematic diagram of activin type II receptor Fc-fusion proteins used as decoy receptors. (B and C) (CAGA12)-luciferase HEK293T293 cells transfected with ALK4-ST or ALK7-ST receptor constructs and treated with ActA (0.62nM), ActE conditioned media (20x), ActC (0.62nM), and 10μM SB-431542 in the presence of increasing amounts (6.25, 12.5, 25 nM) of either ActRIIA-Fc (****P < 0.0001) (****P < 0.0001) (****P = 0.0002) (B) or ActRIIB-Fc (****P < 0.0001) (P = 0.3132) (P = 0.0549) (ns = not significant) (C). (D) Schematic representation of neutralizing antibodies targeting the extracellular domains (ECDs) of the activin type II receptors. (E) (CAGA12)-luciferase HEK293T293 cells either untransfected or transfected with the ALK7-ST type I receptor and treated with ActA (0.62nM), ActE conditioned media (20x), ActC (0.62nM), and 10μM SB-431542 in the presence or absence of neutralizing antibodies targeting ActRIIA, ActRIIB, or both (2.5μg/mL) (****P < 0.0001) (****P < 0.0001) (****P < 0.0001) (ns = not significant) . In (B, C, and E) each point represents a technical replicate within triplicate experiments with bars displaying the mean ± SD. Data are represented as percentages of uninhibited signal (B, C, and E).
Fig 3: Activin E signals via the activin type II receptors.(A) Schematic diagram of activin type II receptor Fc-fusion proteins used as decoy receptors. (B and C) (CAGA12)-luciferase HEK293T293 cells transfected with ALK4-ST or ALK7-ST receptor constructs and treated with ActA (0.62 nM), ActE conditioned media (20×), ActC (0.62 nM), and 10 μM SB-431542 in the presence of increasing amounts (6.25, 12.5, 25 nM) of either ActRIIA-Fc (****P < 0.0001) (****P < 0.0001) (****P = 0.0002) (B) or ActRIIB-Fc (****P < 0.0001) (P = 0.3132) (P = 0.0549) (ns = not significant) (C). (D) Schematic representation of neutralizing antibodies targeting the extracellular domains (ECDs) of the activin type II receptors. (E) (CAGA12)-luciferase HEK293T293 cells either untransfected or transfected with the ALK7-ST type I receptor and treated with ActA (0.62 nM), ActE conditioned media (20×), ActC (0.62 nM), and 10 μM SB-431542 in the presence or absence of neutralizing antibodies targeting ActRIIA, ActRIIB, or both (2.5 µg/ml) (****P < 0.0001) (****P < 0.0001) (****P < 0.0001) (ns = not significant). In (B, C, and E) each point represents a technical replicate within triplicate experiments with bars displaying the mean ± SD. Data are represented as percentages of uninhibited signal (B, C, and E).
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