Western blot
Active GDF3 levels were determined in fresh tissue samples not part of the plasma screen (Additional file 1: Table S3). Hippocampal samples were a random subset picked blindly from the same donors as the cortical samples. All tissues or cells were lysed in RIPA buffer and total protein concentrations were determined with a BCA Protein Assay Kit (Thermo Scientific, Waltham, MA). 10–20 μg of total protein was loaded for each sample into pre-cast 4–12 % bis-tris gels and run with MOPS buffer (Invitrogen, Carlsbad, CA). Gels were transferred onto PVDF membranes (Millipore, Billerica, MA). Antigen specific primary antibodies were incubated overnight at 4 °C and detected with species-specific horseradish-peroxidase labeled secondary antibodies. An ECL Western Blotting Detection kit (GE Healthcare, Cleveland, OH) was used to obtain a chemiluminescence signal, which was detected using Amersham Hyperfilm ECL (GE Healthcare). Band quantification was performed using ImageJ software (version 1.46; NIH, Bethesda, MD). Bands of interest were normalized to actin or neuron specific enolase for a loading control. For active GDF3 we used anti-GDF3 antibodies from Novus Biologicals (Littleton, CO; NBP1-96508).