Enhanced chemiluminescence plus system

Western immunoblotting was carried out as previously described (Silasi et al., 2004 (link); Kovalchuk et al., 2016a (link),b (link),c (link)). In brief, hippocampal 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. The membranes were incubated with primary antibodies against 4-HNE, AKT 1, NPAS4 (1:1,000, Abcam), ERK1/2, FOSB, PCNA (1:1,000, Cell Signaling), and actin (1:2,000, 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 normalized relative to actin or Coomassie staining.

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.

Muscle tissues were lysed in TNT lysis buffer (50 mM Tris [pH 7.5], 75 mM NaCl, and 1% Triton X-100 plus complete protease inhibitor cocktail [Roche]) and clarified by centrifugation. Protein extracts were quantified with a BCA assay (Pierce), normalized, separated by SDS PAGE, transferred to a polyvinylidene difluoride membrane (Millipore) and blotted using a rabbit monoclonal ANTXR1 antibody (clone 37), followed by an HRP-anti-rabbit secondary (Jackson Immunoresearch). Proteins were visualized using the enhanced chemiluminescence plus system (Amersham) according to the supplier’s instructions.

Western immunoblotting was conducted as described previously [50 (link)]. The membranes were incubated with primary antibodies against APE1, OGG1, DNMT1, DNMT3a, MeCP2 (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.

Western blotting was carried out essentially as described previously [23 ]. A total of 30 μg of protein were separated by 12% SDS-PAGE and proteins subsequently were transferred to 0.2 μM nitrocellulose membranes by a wet transfer system. The membranes were blocked with 5% nonfat skim milk in 1x TBS for 1 hr. After blocking, the membranes were incubated with either a 1 : 3000 dilution of a mouse monoclonal anti-human haptoglobin antibody (sc-365396, Santa Cruz Biotechnology Inc., Santa Cruz, CA) for 2 hrs and subsequently incubated with a 1 : 8000 dilution of a rabbit anti-mouse IgG secondary antibody conjugated with horseradish peroxidase (A9044, Sigma, Sigma-Aldrich, St Louis, MO) for 1 hr or with a 1 : 8000 dilution of a polyclonal rabbit anti-human apolipoprotein A-I antibody (sc-30089, Santa Cruz Biotechnology Inc.) for 2 hrs followed by a 1 : 4000 dilution of a HRP-conjugated goat anti-rabbit IgG polyclonal antibody (Pierce Biotechnology, USA) for 1 hr. Protein expression signal was developed using the Enhanced Chemiluminescence Plus system (Amersham Biosciences).