0.22 mm polyvinylidene fluoride membranes

Cells were harvested and lysed with Western/IP lysis buffer (Beyotime, Haimen, China) containing the protease inhibitor phenylmethanesulfonylfluoride fluoride (Beyotime). The protein concentration was determined using the Enhanced BCA Protein Assay Kit (Beyotime). Proteins were denatured at 100 °C for 10 min, subjected to sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS-PAGE), and transferred to 0.22 mm polyvinylidene fluoride membranes (Merck-Millipore, Darmstadt, Germany). The membranes were blocked using 5% non-fat milk and 1% Tween 20 in phosphate-buffered saline (PBS) for 1 h and incubated with primary antibodies at 4 °C overnight. The membranes were subsequently washed and incubated with the appropriate secondary antibodies at room temperature. Finally, the signals were detected by standard analysis of HRPO-induced chemiluminescence. Details of the antibodies used in western blot are listed in Supplementary Table 1.

Cells and exosomes were lysed with western/IP lysis buffer (Beyotime, Haimen, China), and tissues were lysed with radioimmunoprecipitation buffer extraction reagent (Solarbio, Beijing, China); both were supplied with a protease inhibitor cocktail (Roche Applied Science, Indianapolis, IN, USA). The whole-cell lysates were centrifuged at 12000 rpm for 15 min at 4 °C to extract proteins. Proteins were transferred to 0.22 mm polyvinylidene fluoride membranes (Merck-Millipore, Darmstadt, Germany) followed by separation with sodium dodecyl sulfate polyacrylamide gel electrophoresis, and the membranes were blocked with 5% nonfat milk and 1% Tween 20 in PBS for 1 h. Membranes were then incubated with primary antibodies at 4 °C overnight, which are listed as follows: anti-GAPDH (CST, #5174), anti-MELK (Abcam, #ab108529), anti-caspase substrates (CST, #8698), anti-cleaved caspase-3 (CST, #9664), anti-cleaved PARP (CST, #5625), anti-CD9 (CST, #13403), and anti-TSG101 (Abcam, #ab125011). The blots were then washed with PBS with 1% Tween 20 and were incubated with secondary antibodies conjugated to horseradish peroxidase (CST, #7074 or #7076). The signals were detected using HRP-based chemiluminescence analysis.