Total protein was extracted from H9c2 cells and MSCs-exosomes by
radio-immunoprecipitation assay (RIPA) lysis buffer (TaKaRa, Japan) with
protease inhibitors (Roche, China). The concentrations of proteins were detected
by a bicinchoninic acid (BCA) protein assay kit (Pierce, Netherlands). Sodium
dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) was used to
separate equal quantities of total proteins (20 μg per lane), and then separated
proteins were transferred onto polyvinylidene difluoride membrane (Millipore,
MA, USA). Membranes were blocked by PBS-5% fat-free dried milk at room
temperature for 1 h and then incubated at 4 ℃ overnight with anti-CD63
(1:1,000), anti-YAP (1:2,000), anti-phosphor (p)-YAP (1:2,000), anti-tafazzin
(TAZ) (1:2,000), anti-caspase 3 (1:1,000), anti-B-cell lymphoma-2 (Bcl-2)
(1:2,000), anti-Bax (1:1,000), and anti- glyceraldehyde-3-phosphate
dehydrogenase (GAPDH) (1:3,000) (Abcam, UK). Then, the goat anti-rabbit
horseradish peroxidase-conjugated secondary antibody was incubated with
membranes for 2 h. Protein bands were visualized by ChemiDoc XRS+ system
(Bio-Rad, CA, USA).
radio-immunoprecipitation assay (RIPA) lysis buffer (TaKaRa, Japan) with
protease inhibitors (Roche, China). The concentrations of proteins were detected
by a bicinchoninic acid (BCA) protein assay kit (Pierce, Netherlands). Sodium
dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) was used to
separate equal quantities of total proteins (20 μg per lane), and then separated
proteins were transferred onto polyvinylidene difluoride membrane (Millipore,
MA, USA). Membranes were blocked by PBS-5% fat-free dried milk at room
temperature for 1 h and then incubated at 4 ℃ overnight with anti-CD63
(1:1,000), anti-YAP (1:2,000), anti-phosphor (p)-YAP (1:2,000), anti-tafazzin
(TAZ) (1:2,000), anti-caspase 3 (1:1,000), anti-B-cell lymphoma-2 (Bcl-2)
(1:2,000), anti-Bax (1:1,000), and anti- glyceraldehyde-3-phosphate
dehydrogenase (GAPDH) (1:3,000) (Abcam, UK). Then, the goat anti-rabbit
horseradish peroxidase-conjugated secondary antibody was incubated with
membranes for 2 h. Protein bands were visualized by ChemiDoc XRS+ system
(Bio-Rad, CA, USA).