Whole hippocampi were used to assess protein expression. Protein concentration was determined using Bio-Rad Protein Assay Dye (Hercules, California, USA; Cat: 5000006). Samples were mixed with SDS sample buffer and 10 μ of protein was loaded in duplicate on SDS-PAGE gels, then transferred to PVDF Transfer Membrane (Millipore Sigma, Darmstadt, Germany). Membranes were blocked using 5% milk for 1–2 hours. Antibodies were diluted in 1% Tween in PBS or 5% milk prepared in 1% Tween in PBS and incubated overnight at 4°C. Membranes were then washed and incubated with secondary antibody, either Rabbit IgG HRP Linked Whole Antibody (Millipore Sigma, Darmstadt, Germany; Cat: GENA934) or Mouse IgG HRP Linked Whole Antibody (Millipore Sigma, Darmstadt, Germany; Cat: NXA931V). Signals were detected with enhanced chemiluminescence using Pierce ECL Blotting Substrate (Thermo Scientific, Carlsbad, CA, USA, Cat:32106). If a second detection was needed, blots were stripped using Restore Western Blot Stripping Buffer (Thermo Scientific, Carlsbad, CA, USA, Cat:21059), blocked again in 5% milk, and incubated overnight with the desired antibody.
Signal intensities of proteins were normalized to GAPDH signal on the same blot. Duplicates were averaged for each data point. Protein-specific signals on Western blots were quantified densitometrically using NIH ImageJ software (Bethesda, Maryland, USA).
Signal intensities of proteins were normalized to GAPDH signal on the same blot. Duplicates were averaged for each data point. Protein-specific signals on Western blots were quantified densitometrically using NIH ImageJ software (Bethesda, Maryland, USA).