Immunoblot Analysis of Plasma GDF11

Immunoblot analysis of the plasma GDF11 levels was performed as described previously.^18,19 The plasma samples were diluted with saline and the diluted samples were mixed with sample buffer (Bio-Rad Laboratories, Hercules, CA). The samples containing 50 µg of protein were loaded and separated by electrophoresis on 10% SDS polyacrylamide gels. In addition to the samples, in order to compare samples from separate gels, 50 µg of a particular sample was loaded. After electrophoresis, the separated proteins were transferred to a PVDF membrane (Merck Millipore Ltd., Darmstadt, Germany). We used an antibody raised against GDF11 specifically (1:1000 dilution, R&D Systems Inc., Minneapolis, MN).¹⁸ Bound antibodies were visualized using a peroxidase-conjugated anti-mouse goat antibody (1:2000 dilution, Santa Cruz Biotechnology Inc., Dallas, TX) and enhanced chemiluminescence (GE Healthcare Ltd., Buckinghamshire, UK) with a chemiluminescence imaging system (LAS-4000 mini; Fujifilm CO., Tokyo, Japan). Ponceau S staining was used to evaluate the amounts of protein. The GDF11 levels were calculated by measuring the intensity of the bands. Band intensity was quantified using ImageJ 1.52v software (National Institutes of Health, Bethesda, MD). In order to compare across blots, the protein levels were normalized against a particular sample.