Ecl developing solution

We quantified GASF-derived exosomes proteins using a bicinchoninic acid Protein Assay Kit (Beyotime Biotechnology Inc., Shanghai, China) after lysing with exosomes dedicated lysate (UR33101, Shanghai Umibio Co., Ltd., Shanghai, China). Prior to loading onto 10% SDS-polyacrylamide gels, these protein samples were boiled in loading buffer for 10 minutes at 100°C. During the electrophoresis, the samples were run at 80 V (30 minutes) and 120 V (60 minutes). Then, the proteins were transferred onto PVDF membranes using a transfer equipment of wet means operating at 200 mA for 120 minutes. After blocking for at least 1 hour at room temperature with the block solution (containing 5% skimmed milk powder and 0.1% Tween 20) on a shaker, these membranes were rinsed with distilled water. Primary antibodies, TSG101, CD63, and CD81 from Abcam all with a 1:1000 dilution were then added to the membranes overnight at 4°C. On Day 2, a TBST wash was performed 3 times, in each instance for 10 minutes. Incubation with secondary antibodies (1:1000 goat anti-mouse IgG) was carried out on the PVDF membranes for 1 hour at room temperature and analyzed by the Bio-Rad Protein Imaging System using ECL developing solution (Thermo Scientific, Rockford, IL).

A human normal biliary epithelial cell line (HIBE) and human cholangiocarcinoma cell lines (RBE, QBC939, HuH28) were purchased from ATCC. qRT-PCR and reverse transcription kits were purchased from TransGen Biotech, Beijing, China. CCK-8 assay kits were purchased from Promega, USA. Transwell kits were purchased from Shanghai Yantuo Biotechnology Co., Ltd. Phosphate buffer solution (PBS) and fetal bovine serum (FBS) were purchased from Gibco, Rockville, MD, USA. Trizol reagents were purchased from Beijing Biolab Science and Technology Co., Ltd. Dual-luciferase reporter gene assay (DLRGA) kits were purchased from Beijing Biolab Science and Technology Co., Ltd. Bax, Bcl-2, Caspase-3, and β-Actin antibodies were purchased from Cell Signaling Technology, Boston, Massachusetts, USA. Goat anti-rabbit IgG secondary antibody was purchased from Thermo Fisher Scientific, Shanghai. RIPA and BCA protein assay kits were purchased from Thermo Fisher Scientific, Waltham, MA, USA. An ECL developing solution was purchased from Thermo Fisher Scientific, Waltham, MA, USA. All primers were designed and synthesized by Sangon Biotech (Shanghai) Co., Ltd.