Bradford protein quantitative kit

The 12 samples were ground individually in liquid nitrogen and lysed with lysis buffer containing 100 mM NH₄HCO₃ (pH = 8), 8 M urea, and 0.2% SDS, followed by 5 min of ultrasonication on ice. The lysate was centrifuged at 12,000× g for 15 min at 4 °C, and the supernatant was transferred to a clean tube. Extracts from each sample were reduced with 10 mM DTT for 1 h at 56 °C and subsequently alkylated with sufficient iodoacetamide for 1 h at room temperature in the dark. Then, samples were completely mixed with 4 volumes of precooled acetone by vortexing and incubated at −20 °C for at least 2 h. Samples were then centrifuged, and the precipitation was collected. After washing twice with cold acetone, the pellet was dissolved by dissolution buffer containing 0.1 M triethylammonium bicarbonate (TEAB, pH = 8.5) and 6 M urea.
BSA standard protein solution was prepared according to the instructions of the Bradford Protein Quantitative Kit (Beyotime, Shanghai, China), with concentrations ranging from 0 to 0.5 g/L; this allowed us to determine the protein concentrations of individual samples.

CSF specimens were thawed and transferred into a 1.5 mL centrifuge tube and lysed with DB lysis buffer [8 M urea (10023218, Sinopharm) and 100 mM triethylammonium bicarbonate (TEAB; T7408-500ML, Sigma, pH 8.5)]. The lysate was centrifuged at 12,000 × g for 15 minutes at 4 °C. The supernatant was deoxidized using 10 mM DL-dithiothreitol (D9163-25G, Sigma) for 1 hour at 56 °C, followed by alkylation with adequate iodoacetamide (I6125-25G, Sigma) for 1 hour at room temperature in the dark.
A linear protein concentration ranging from 0 to 0.5 g/L was prepared as bovine serum albumin (BSA) standard protein solution following the Bradford protein quantitative kit (P0006, Beyotime) manual. The sample solutions with gradient dilutions and BSA standard protein solutions in a 20 μL volume were placed into a 96-well plate. The plate was filled with 180 μL of G250 dye solution immediately and stored at room temperature, and the absorbance of each well was determined at 595 nm after 5 minutes of incubation. The absorbance of the standard protein solution was used to create the standard curve, and subsequently, the concentration of the protein sample was calculated. Each assay was conducted in triplicate. Equal amounts of the protein sample (20 μg) were separated by 12% SDS‒PAGE. The gel was dyed with Coomassie brilliant blue R-250 and decolored until the bands were visualized clearly.

Dulbecco's modified Eagle's medium (DMEM) was obtained from Biological Industries Israel Beit-Haemek Ltd, Kibbutz Beit-Haemek, Israel. Heat-inactivated horse serum (hiHS) was purchased from Thermo Fisher Scientific, Auckland, New Zealand. Minimal essential medium (MEM), opti-MEM medium, Neurobasal medium, B27 supplements, Rhodamine 123, Lipofectamine RNAiMAX reagent, TRIzol reagent, RevertAid reverse transcriptase, dNTP and RiboLock RNase inhibitor were purchased from Thermo Fisher Scientific, Grand Island, NY, USA. Fetal calf serum (FCS) was obtained from Hangzhou Tianhang Biological Technology Co., Ltd, Hangzhou, China. STS, caspase 3 activity assay kit, Bradford protein quantitative kit, and bicinchoninic acid (BCA) protein assay kit were bought from Beyotime Institute of Biotechnology, Haimen, Jiangsu, China. pEGFP-C3 was obtained from Clontech (Mountain View, CA, USA). Pan-caspase inhibitor z-VAD-fmk (CasI) and caspase 3-specific inhibitor Ac-DEVD-CHO (Cas3I) were purchased from EMD Biosciences, Inc., San Diego, CA, USA. Poly-d-lysine, cytosine β-D-arabinofuranoside (AraC), spermidine and CQ were bought from Sigma-Aldrich (Shanghai) Trading Co., Ltd, Shanghai, China. Rapa and BafA1 were obtained from Alexis Biochemicals, San Diego, CA, USA.