Immunoblotting Analysis of miRNA Machinery

Western immunoblotting was conducted as previously described [114 (link)-117 (link)]. In brief, around 50 mg of PFC tissues were sonicated in ice-cold 1% SDS and immediately boiled. Protein concentrations were determined using the Bradford assay (BioRad, Hercules, CA). Equal amounts of protein (10-30 μg) were separated by SDS-PAGE into slab gels of 10-15% polyacrylamide and transferred to polyvinylidene difluoride membranes (Amersham Biosciences, Baie d’Urfé, Quebec). Eight membranes were prepared in total. Due to scarce amount of tissues, membranes were re-used and re-probed to allow for analysis of miRNA machinery and targets (this study), as well as epigenetic regulators (Kovalchuk et al., 2017, Aging, in press). The membranes were incubated with primary antibodies against BDNF, Dicer and Ago2 (1:1000, Abcam), and actin (1:2000, Abcam) overnight at 4° C. Primary antibody binding was detected using horseradish peroxidase-conjugated secondary antibodies and the Enhanced Chemiluminescence Plus System (Amersham Biosciences, Baie d’Urfé, Quebec). Chemiluminescence was detected using a FluorChem HD2 camera with FluorChem software (Cell Biosciences). The membranes were stained with Coomassie blue (BioRad, Hercules, CA) to confirm equal protein loading. Signals were quantified using NIH Image J64 software and normalised relative to actin or Coomassie staining.