Rabbit anti cd63

The samples used for identification of widely expressed protein markers in EVs and quantification of caspase 3 and cleaved-caspase 3 expression in three groups (Blank, Dox, EV) were suspended in 100 μl radio-immunoprecipitation assay (RIPA) buffer (Solarbio, Shanghai, China), the protein of which (30 μg) was subjected to 10% SDS-PAGE and transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, Darmstadt, Germany). After being immersed in 5% nonfat milk for 2 h, they were incubated with primary antibodies overnight at 4 °C and secondary antibodies for 2 h at room temperature. The signal was detected by the Pierce enhanced chemiluminescence western blotting substrate (Millipore). The primary antibodies used for western blotting analysis were as follows: rabbit anti-Alix (Wanleibio, Shengyang, China), rabbit anti-CD9 (Abcam, Cambridge, UK), rabbit anti-CD63 (Wanleibio, Shengyang, China), rabbit anti-TSG101 (Abcam, Cambridge, UK), rabbit anti-caspase 3 (Wanleibio, Shengyang, China), rabbit anti-cleaved-caspase 3 (Wanleibio, Shengyang, China), and mouse anti-tubulin (Abcam, Cambridge, UK). Relevant experimental operations were following the manufacturer’s instructions.

Cells were harvested in RIPA lysis buffer (Solarbio, Shanghai, China), quantified by a BCA Protein Assay Kit (Promega), separated by 10% SDS-PAGE and transferred to polyvinylidene fluoride membranes (Millipore, Darmstadt, Germany). After blocking with 5% skim milk for 2 h, the membranes were incubated with primary antibodies at 4 °C overnight. After three times washing with TBST, the membranes were incubated with secondary antibodies for 2 h at room temperature. The Pierce enhanced chemiluminescence western blotting substrate (Millipore) was used to detect the signal. The primary antibodies were used for western blot analysis: rabbit anti-Sox2 (Santa Cruz Biotechnology), rabbit anti-Oct4 (Santa Cruz Biotechnology), rabbit anti-Nanog (Bethyl), rabbit anti-P53 (Santa Cruz Biotechnology), rabbit anti-P16 (Wanleibio, Shenyang, China), rabbit anti-CD9 (Abcam, Cambridge, UK), rabbit anti-CD63 (Wanleibio, Shenyang, China), rabbit anti-IGF1R (Novus biological), mouse ant-IGF1 (Novus biological).