Pvdf membrane

The mouse brain hippocampus tissues were homogenized in RIPA lysis buffer (#89900, Thermo Fisher Scientific, USA) containing protease inhibitor (Servicebio, China) on ice for 30 min and centrifuged at 14000 × g for 20 min at 4°C. The experiment was performed according to our previous experimental steps [26 (link)]. The supernatants were gathered and applied to the BCA assay (#P0010, Beyotime, China) to judge protein concentration and then separated by 10% and 12% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) followed by transferring the proteins from the gels to polyvinylidene difluoride (PVDF) membrane (Millipore, Germany) and blocking the membranes with 5% skim milk. The membranes were exposed to the primary antibodies for an extended period at a temperature of 4°C: anti‐PSD95 (1:1000, #3409, CST), anti‐Parkin (1:1000, #4211, CST), anti‐LC3A/B (1:1000, #12741, CST), anti‐SQSTM1/P62 (1:1000, #5114, CST), anti‐Beclin1(1:1000, #3495, CST), anti‐GAPDH(1:5000,#60, 004‐1. Proteintech) and β‐actin (1:5000, #380624, ZEN BIO). Following a thorough wash with 1× TBST and a subsequent incubation with secondary antibodies for an hour, the membranes were treated with a fluorescent solution. Images were obtained using the ChemiDoc MP Imaging System (Bio‐Rad, USA). The densities of the bands were quantified by using ImageJ, while graphs were generated by GraphPad Prism 8 software.

To quantify proteins, tissues containing the mPFC, NAc, CPu, and hippocampus were isolated from animals and immediately flash‐frozen on dry ice. The frozen tissue was then lysed in SDS lysis buffer, followed by centrifugation at 12 000 rpm at 4 °C for 15 min to collect the supernatant. Protein concentration in the samples was determined using the Omni‐Easy Instant BCA Protein Assay Kit (Epizyme, ZJ102). Samples were diluted in sample buffer, and was loaded on 10% polyacrylamide gels supplemented with β‐mercaptoethanol and bromophenol blue. The gels were transferred onto polyvinylidene difluoride (PVDF) membranes (Millipore) for 2 h on ice. The membranes were then blocked in 5% milk in TBST (20 mM Tris pH 7.5, 150 mM NaCl, 0.1% Tween‐20) for 1 h at room temperature. Subsequently, the membranes were incubated overnight at 4 °C with the primary antibodies. After washing the membranes in 5% TBST, they were incubated with the secondary antibody (1:10 000) at room temperature. Goat anti‐Rabbit IgG (PIONEER Biotechnology, 31 460) secondary antibodies was used. Following the antibody incubation, the protein bands were visualized using enhanced chemiluminescence (ECL) with the SuperSignal West Pico chemiluminescent substrate (Thermo Fisher Scientific). The primary antibodies were shown as follows: KDM7A (Genetex, GTX32688, 1:500), FSCN1 (Proteintech, 14384‐1‐AP, 1:5000), and GAPDH (Abcam, ab263962, 1:1000).