Ab13847

Total protein was extracted with the use of radio‐Immunoprecipitation Assay lysate buffer (R0010; Beijing Solarbio Science & Technology Co., Ltd. Beijing, China). Protein concentration of each sample was determined by using a BCA kit (GBCBIO Technologies, Guangzhou, Guangdong, China). The protein was separated by polyacrylamide gel electrophoresis, transferred to a polyvinylidene fluoride membrane (Millipore) and sealed with 5% BSA at room temperature for 1 h. Primary rabbit antibodies (1:1000) from Abcam to cleaved‐Caspase3 (ab49822), Caspase3 (ab13847), B cell lymphoma‐2 (Bcl‐2; ab196495), Bcl‐2‐associated X (Bax; ab32503), FOS (ab190289) and p65 (ab16502) as well as primary rabbit antibody to p‐p65 (Ser536) (1:1000; 3033, Cell Signaling Technology) were added to the membrane and incubated overnight. The following day, the membrane was incubated with goat anti‐rabbit IgG (ab97051, 1:2000, Abcam) at room temperature. The immunocomplexes on the membrane were visualized using enhanced chemiluminescence reagent and imaged using Image Quant LAS 4000C gel imager (GE, General Electric Company, Schenectady, NY, USA). With rabbit anti‐β‐actin (1:3000; Abcam, ab8227) serving as an internal reference, the grey value of each band was analysed by gel image analysis software Image J.

Total protein was isolated using RIPA lysis buffer, separated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis, and then transferred onto polyvinylidene fluoride membranes. The membrane was and probed with diluted primary rabbit antibodies against Caspase-3 (ab13847), Bcl-2-associated X protein (Bax; ab32503), B-cell lymphoma 2 (Bcl-2) (ab32124), Bcl-XL (ab32370), TGF-β1 (ab92486), β-actin (ab8227), GAPDH (ab181602), E-cadherin (3195), N-cadherin (13116), matrix metalloproteinase (MMP)3 (14351), MMP9 (13667), Smad2 (5339), Smad3 (9523), phosphorylated (p)-Smad2 (18338), and p-Smad3 (9520, Cell Signaling Technologies) overnight at 4°C. The antibodies were from Abcam except for p-Smad2/3. After washing, membrane was re-probed with the horseradish peroxidase-conjugated goat anti-rabbit secondary antibody (ab205719, 1: 2000, Abcam) for 1 h. The protein bands were visualized using enhanced chemiluminescence (EMD Millipore). β-actin and GAPDH were used as internal controls. The gray values were analyzed with Image J software.

Total protein of ex vivo cells, exosomes, and hippocampus tissue were extracted using radioimmunoprecipitation assay (RIPA) solution lysis buffer (Beyotime, Shanghai, China). The concentration was evaluated by bicinchoninic acid (BCA) kits, and then 30 μg protein was separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The separated protein was then transferred to the activated polyvinylidene difluoride (PVDF). The membrane was blocked with 5% non-fat blocking grade milk (Bio-Rad, Hercules, CA, USA) and subsequently incubated with the following primary antibodies against CD81 (Abcam, Ab109201), CD9 (Abcam, Ab92726), CD63 (Affinity, AF5117), CD80 (Abcam, Ab215166), CD68 (Affinity, AF7518), CD206 (Abcam, Ab64693), CD163 (Abcam, Ab182433), EpCAM (Affinity, DF6311), BAX (Cell signaling, 2772), Bcl-2 (Abcam, Ab59348), Caspase 3 (Abcam, Ab13847), and LC3 (Cell signaling, 4108). Then, the membrane was incubated with appropriate horseradish peroxidase-conjugated secondary antibodies for 1 h at room temperature. Subsequently, the blots were visualized by Versa Doc (Bio-Rad Laboratories, Inc.). Glyceraldehyde phosphate dehydrogenase (GAPDH) (AB-P-R 001) served as the internal control.