(A) Affinity of GDF11 SOMAmer for recombinant GDF11 and myostatin. Binding of the GDF11 SOMAmer to GDF11 (shown in blue) and myostatin (shown in red) proteins as measured by dissociation-enhanced lanthanide fluorescent immunoassay (DELFIA). Data represent means ± SD from three technical replicates.

(B) Western blot analysis to determine specificity of Abcam antibody to GDF11 versus myostatin (GDF8). An anti-GDF11 antibody from Abcam was tested for specificity using a concentration gradient of recombinant GDF11 and myostatin, ranging from 6.25 ng to 100 ng, and was found to cross-react with myostatin. Even though this is a denaturing gel, bands consistent with dimer and even high molecular forms consistent with aggregates of the recombinant material are evident.

(C) Western analysis on sera from young and old mice. Sera samples from four different young animals (4 months old; 1, 2, 3, 4) and four different old animals (23 months old; 5, 6, 7, 8) were tested by western analysis for myostatin/GDF11 levels (top). Coomasie staining (bottom) demonstrates equivalent loading of each lane. Lane with ladder is indicated. The dimer band was not fully denatured to monomer. There was an increase in GDF11/myostatin dimer levels in the sera from older animals in comparison to young animals. Densitometry of monomer + dimer is provided on the right, indicating an overall increase in myostatin/GDF11 levels in the mouse sera (∗p < 0.05).

(D) GDF11 and myostatin mRNA content in in skeletal muscles of Sprague-Dawley male rats aged 6, 12, 18, 21, and 24 months (data derived from the RNA-seq analyses). RNA-seq analysis demonstrates that GDF11 expression increases as a function of age (comparing mRNA obtained from muscles from 6-, 12-, 18-, 21-, and 24-month-old rats). In contrast, myostatin (MSTN) expression decreases with age in rats. The y axis is the standardized expression level, with mean of 0 and standard deviation of 1. GOF, goodness of fit to a sigmoidal curve; FC, fold change between 24 m and 6 m.

(E) GDF11 protein levels in sera from young and old rats determined by immunoassay. GDF11 protein content in serum from young (6 months) or old (24 months) rats was measured by immunoassay. Old rats had higher levels of GDF11 compared with young. Data are mean ± SEM (p = 0.0534, Student’s t test).

(F) GDF11 protein levels in sera from young and older humans determined by immunoassay. GDF11 protein content was measured in serum samples from nine older (aged >60 years, males, shown in red) or ten young (aged 20-30 years, males, shown in blue). The median GDF11 concentration in serum of older humans was higher than in younger humans, but this did not reach statistical significance. Serum samples from three young and one old subject had GDF11 below a detection limit (less than 0.274 ng/ml), shown with a dotted horizontal line.