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Fig. 6 | BMC Biology

Fig. 6

From: Structural basis for potency differences between GDF8 and GDF11

Fig. 6

Enhanced type I receptor utilization by GDF11 compared to GDF8. a, b, Luciferase reporter assays showing the inhibitory activity following titration of ActRIIB-ECD (a), and Fc-ActRIIB-ECD (b) against a constant concentration (0.62 nM, dashed bar) of GDF8 (blue) or GDF11 (orange) in HEK293 (CAGA)12 cells. Refer to Table 3 for a corresponding analysis of the inhibition curves. c Differences in type I receptor utilization by GDF11 following transient transfection of a single type I receptor (ALK4, ALK5, or ALK7) or a combination of type I receptors (ALK4/ALK5, ALK4/ALK7, ALK5/ALK7, or ALK4/ALK5/ALK7). RIB L17 cells were co-transfected with 1.25 ng of the individual receptor DNA alone or various receptor DNA combinations (1.25 ng each receptor) and 2.5 ng (CAGA)12 promoter plasmid driving the luciferase gene. Purified recombinant ligands were added 12 h post transfection. Cells were then lysed and assessed for luciferase activity 8 h post ligand treatment. d Treatment of RIB L17 cells with DMSO or the type I receptor small molecule inhibitor SB431542 following transfection of empty vector (EV) or ALK7 S270T. RIB L17 cells were co-transfected with 1.25 ng of the individual receptor DNA and 2.5 ng (CAGA)12 promoter plasmid driving the luciferase gene. Purified recombinant ligands were added 12 h post transfection. Cells were then lysed and assessed for luciferase activity 8 h post ligand treatment. Data information: In a, b, data are presented as fraction activation (ligand response at antagonist concentration/ligand response at 0 nM antagonist). Each concentration was performed in triplicate and shown as the mean ± SEM of two independent experiments. Data from independent experiments were combined and fit to non-linear regression with a variable slope. In c, d, data are presented as fold activation defined as the total activation from each ligand compared to cells only transfected with the (CAGA)12 reporter construct. Each bar is the mean ± SEM. A representative experiment is shown of at least two independent experiments in which each concentration was performed in duplicate or triplicate. Only comparisons between GDF8 and GDF11 were made. *P ≤ 0.05 and **P ≤ 0.001 (Student’s t test). Ligand sources: GDF8 and GDF11, gift from Acceleron Pharma; Activin A, Activin B, and TGFβ3, produced and purified as described in “Methods

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