Fluorchem hd2 camera

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.

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.

Protein was extracted by sonication of cells in 100 µL cold 1% SDS containing protease inhibitor (Roche). 40 µg of protein was separated by SDS-PAGE in slab gels of 6% (DNMT1) or 10% (MAT2A) polyacrylamide and transferred to Amersham Hybond-P polyvinylidene difluoride (PVDF) membranes (Amersham Biosciences). The membranes were incubated with primary antibodies overnight at 4°C (DNMT1 [Abcam], MAT2A [Abcam], GAPDH [Santa Cruz]). Antibody binding was revealed by incubation with horseradish peroxidase-conjugated secondary antibodies followed by ECL Plus immuno-blotting detection system (Amersham Biosciences). Chemiluminescence was detected using a FluorChem HD2 camera with FluorChem software (Cell Biosciences). The signals were quantified using NIH Image J64 software and normalized relative to GAPDH.