Protein extraction.
For protein analysis, we used a previous published protocol of protein
extraction (Lorente et al., 2022). 0.5 mL of cold lysis buffer (1%
IGEPAL CA-630, 20mMTris-HCl pH 8, 130MNaCl, 10mMNaF, and 1% proteases
inhibitor cocktail [Sigma, Darmstad, Germany]) was used to
homogenate 250 mg of brain tissue, thus the volume of the lysis buffer
was adapted to the real quantity of tissue in each sample. Then we
maintained the samples in ice for 30 minutes, before centrifugate at
13000 G for 15 minutes at 4 ยบ C and collect the supernatant.
Furthermore, we determined the concentration of the lysates by using a
Bradford protein assay kit (Bio-Rad).