Mouse anti bax

Mouse anti-Bax, mouse anti-Bcl-2, rabbit anti-Caspase-3, rabbit anti-Bid, rabbit anti-PARP, rabbit anti-Survivin, and rabbit anti-GAPDH were obtained from Proteintech (USA). Horseradish peroxidase- (HRP-) conjugated goat anti-mouse and anti-rabbit IgG antibodies were provided by Beijing Zhongshan Golden Bridge Biotechnology Co. Ltd. (Beijing, China). Protein assay kit was obtained from Beijing Pulilai Gene Technology Co., Ltd. (Beijing, China). Reverse transcription kit was obtained from Promega (USA).

The isolated tissues, cells, or EVs were treated with RIPA lysis buffer supplemented with the Protease Inhibitor Cocktail (Roche) at 4 °C for 30 minutes. The protein concentrations were assessed using the BCA Protein Assay Kit. Subsequently, equal amounts of purified proteins were separated using SDS-PAGE and transferred onto a nitrocellulose membrane in an ice bath. The nitrocellulose membrane was blocked with 5-8% nonfat dried milk for 1 h at room temperature, followed by overnight incubation with primary antibodies at 4 °C. The antibodies employed were mouse anti-CD81 (Abcam, ab79559), mouse anti-TSG101 (Abcam, ab83), rabbit anti-GM130 (Proteintech, 11308-1-AP), mouse anti-BAX (ProteinTech, 60267-1-Ig), rabbit anti-TfR (Proteintech, 10084-2-AP), rabbit anti-caspase-3 (Cell Signaling Technology, #14220), and mouse anti-GAPDH (Proteintech, 60004-1-Ig). The nitrocellulose membrane was subsequently incubated with corresponding secondary antibodies at room temperature for 1 h and visualized using the ECL Prime Western Blotting Detection Reagent (GE, UK).

Tissues were homogenized in RIPA plus buffer containing a cocktail of EDTA-free protease inhibitors. The homogenate was centrifuged at 12,000 rpm for 30 min at 4°C. Protein concentration was assayed using the BCA method, then loaded and subjected to electrophoresis in 10% SDS-PAGE gels, and transferred onto PVDF membranes. The membranes were then blocked in 5% BSA for 2 hour and then incubated with one of the following primary antibodies: rabbit anti-caspase-3 (1 : 500, Proteintech) or mouse anti-Bax (1 : 100, Proteintech) or rabbit anti-Bcl-2 (1 : 500, Proteintech) or mouse anti-Notch1(1 : 500, Santa Cruz) or mouse anti-Hes1 (1 : 1000, Santa Cruz), with gentle shaking at 4°C overnight. Then, horseradish peroxidase conjugated goat anti-mouse IgG (1 : 50000, Proteintech) or goat anti-rabbit IgG (1 : 50000, Proteintech) secondary antibodies were incubated with the membranes for 2 hours at room temperature. The immunopositive bands were visualized using an enhanced chemiluminescent substrate (Thermo Fisher) and Bio-Rad ChemiDoc XRS digital documentation system. The amount of protein expression is presented relative to the levels of β-actin.