Ecl western blot detection reagents

Total proteins were got by cell lysis in ice-cold RIPA buffer in DU145 cells. Cells extracts were subjected to a 10% polyacrylamide gel and blotted onto a polyvinylidene difluoride (PVDF). The blot was then blocked with 5% non-fat milk in TBST for 2 h and subsequently incubated at 4°C overnight with primary antibodies anti-MMP-9 (1:1,000), as well as p-Akt, t-Akt, p-mTOR, mTOR, Bcl-2, Bax, and anti-GAPDH (1:500), and then cultivated with goat anti-rabbit IgG-HRP. The bands were visualized via ECL Western blot detection reagents (Thermo Fisher Scientific, Inc.), and the results were analyzed by Quantity One (Bio-Rad Laboratories, Inc. Hercules, CA, USA) software.

Proteins were extracted using RIPA lysis buffer (cat. no. HY-K1001; MedChemExpress) from JEG-3 cells and MSCs-Ex. The concentration of the isolated protein was quantified via BCA Protein Assay kit (cat. no. 23225; Thermo Fisher Scientific, Inc.). Isolated proteins (5 µg) were mixed with 5X SDS-PAGE protein loading buffer (cat. no. 20315ES05; Shanghai Yeasen Biotechnology Co., Ltd.). Proteins (20 µg) were separated on 12% SDS-acrylamide gels, followed by transferring onto PVDF membranes, which were incubated with 5% non-fat milk for 1 h at room temperature, and with rabbit anti-HIF-1α (1:1,000; cat. no. ab179483; Abcam), rabbit anti-CD9 (1:1,000; cat. no. ab236630; Abcam), rabbit anti-CD81 (1:1,000; cat. no. ab79559; Abcam), rabbit anti-LAMP-2B (1:1,000; cat. no. ab18529; Abcam), rabbit anti-TSG101 (1:1,000; cat. no. ab125011; Abcam) and rabbit anti-GAPDH (1:10,000; cat. no. 10494-1-AP; ProteinTech Group, Inc.) overnight at 4°C. Subsequently, membranes were incubated with horseradish peroxidase-conjugated mouse anti-rabbit IgG (1:5,000; cat. no. BM2006; Boster Biological Technology) for 1 h at room temperature. Protein bands were determined via ECL western blot detection reagents (Thermo Fisher Scientific, Inc.). The protein gray value was calculated using ImageJ (Version 1.5.3; National Institutes of Health).

Protein was harvested from cells with RIPA Lysis Buffer (Beyotime, Shanghai, P.R. China) according to the manufacturer’s instructions at 48 h after transfection. BCA Protein Assay kit (Beyotime) was used to determine protein concentration. Equal amounts of protein samples (10 μg per condition) were prepared in loading buffer and boiled at 100°C for 10 min, and then the boiled samples were separated on 15% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels and transferred to polyvinylidene difluoride (PVDF) membranes (Millipore Corp., Billerica, MA, USA) at 100 V for 1 h. The PVDF membranes were blocked with 5% nonfat milk in phosphate-buffered saline (PBS) for 1 h at room temperature and subjected to anti-Kim-1 primary antibody (Sigma-Aldrich, St. Louis, MO, USA) incubation at 4°C overnight. After washing, membranes were probed with goat anti-mouse secondary antibodies. Immunoreactive proteins were visualized by enhanced chemiluminescence (ECL) Western blot detection reagents (Thermo Fisher Scientific, Waltham, MA, USA). Before each step, the PVDF membranes were washed three times by PBST. Protein quantification was conducted by ImageJ software [National Institutes of Health (NIH), Bethesda, MD, USA]. The gray values of protein were achieved as gray level of protein band/gray level of loading control.