Ab238126

The total protein of tissues and cells was extracted by high-efficiency RIPA lysis buffer (R0010, Solarbio). After centrifugation at 15,000 rpm/min for 15 min and lysis at 4°C for 15 min, the supernatant was harvested, and the protein concentration was evaluated employing the bicinchoninic acid kit (20201ES76, Yeasen Biotechnology, Shanghai, China). Following separation utilizing polyacrylamide gel electrophoresis, the protein was transferred onto PVDF membranes which were sealed at 5% BSA for 1 h at ambient temperature and probed with the diluted primary antirabbit antibodies (all from Abcam) against Sesn2 (ab178518, 1: 10000), Srx1 (ab203613, 1: 10000), Trx1 (ab273877, 1: 10000), Beclin-1 (ab210498, 1: 10000), LC3A/B (ab128025, 1: 1000), Ki67 (ab92742, 1: 10000), Aggrecan (ab3778, 1: 10000), MMP3 (ab39012, 1: 10000), GRP94 (ab238126, 1: 10000), APOB (ab20737, 1: 10000), and GAPDH (ab8245, 1: 10000) overnight at 4°C. The next day, the membrane was reprobed with HRP-labeled goat antirabbit IgG (ab205718, 1: 20000, Abcam) for 1 h at ambient temperature. The developing solution was added for development. ImageJ 1.48 software (National Institutes of Health) was employed for protein quantitative analysis, and protein quantitative analysis was implemented with the gray value ratio of each protein to the internal reference GAPDH.

Antibodies for HSP90B1 (ab238126) were purchased from Abcam company. Samples were embedded in paraffin at a thickness of 4 μm. Deparaffinization and rehydration were performed on each slide. To eliminate aldehyde linkages from antigens, they were reextracted using a pressure cooker and 0.01 M citrate buffer (pH 6). The slides were incubated with HSP90B1 antibody (1: 2000) overnight. After incubating the HRP-labelled secondary antibody for 1 h, immunodetection was performed the following day using diaminobenzidine, according to the manufacturer’s instructions [61 (link)].

Total proteins of the exosomes were extracted with standard RIPA buffer with PMSF (MCE, United States) at a volume ratio of 99:1, and the concentration of protein was normalized using the BCA assay. The protein samples (25 μg) were then subjected to 10% SDS-PAGE and transferred onto a membrane. The PVDF membrane was incubated with primary antibodies at 4°C overnight, including CD9, CD63, TSG101, and GRP94 (Abcam, ab92726, ab134045, ab125011, and ab238126 Cambs, United Kingdom), and then, the membrane was incubated with HRP-conjugated secondary antibodies (Abcam, Cambs, United Kingdom) for 1 h (Min et al., 2019 (link)).