Hrp linked goat anti rabbit igg

The BMSCs, exosomes, and myocardial tissue were subjected to lysis through RIPA lysis buffer® (Millipore Corporation™, Billerica, MA, USA), with the total proteomic concentration in cell exosomes or tissue asserted using a BCA protein assay kit® (Beyotime™, Shanghai, China), following the protocol provided by the manufacturer. Equivalent proteomic content was resolved in 10% SDS-PAGE gels and transferred into PVDF membranes (Merck Millipore™, MA, USA) which were blocked using 5% nonfat milk for 60 min/RT.
The membranes were incubated using primary antibodies against Nrf2 (1 : 1000, Invitrogen), HO-1 (1 : 1000, Abcam, Cambridge, UK), CD9 (1 : 500, Invitrogen), CD63 (1 : 500, Invitrogen), CD81 (1 : 2000, Abcam), TSG101 (1 : 500, Santa Cruz, CA, USA), Alix (1 : 500, Santa Cruz), calnexin (1 : 500, Santa Cruz), or GAPDH (1 : 10000, Abcam) at 4°C overnight, followed by incubation using HRP-linked goat anti-rabbit IgG (1 : 5000, Abcam) at 37°C for 1 hour. Proteomic bands were developed and scrutinized through enhanced chemiluminescence (ECL) (Thermo Scientific™, Rockford, IL, USA), quantified using ImageJ software.

The amounts of total protein from lEV samples were determined with a bicinchoninic acid assay kit (Pierce, Thermo Scientific, Rockford, IL, USA), according to the manufacturer’s instructions. Western blot analysis was used to determine the EV protein markers, cardiomyocyte marker and EV purity in lEV samples. All samples were adjusted to 30 μg of total protein before mixing with reducing sample buffer and loaded into 10% SDS–polyacrylamide gel electrophoresis. Then, proteins were transferred to a PVDF membrane (Amersham Hybond PVDF Membrane, GE Healthcare Life Sciences, Freiburg, Germany). Membrane was blocked with 5% w/v non-fat milk in TBST buffer and then incubated with primary antibodies; anti-CD63, anti-Alix, anti-Apolipoprotein A (Abcam, Cambridge, MA, USA) and anti-Caveolin 3 (Invitrogen, Thermo Fisher Scientific, Waltham, MA USA) overnight at 4 °C. After washing, membranes were incubated with HRP linked goat anti-rabbit IgG (Abcam Cambridge, MA, USA) for 1 h at room temperature. Chemiluminescent detection was performed using Clarity Western ECL Substrate (Biorad Laboratories, Inc, Hercules, CA, USA). Protein bands were visualized by ImageQuant LAS 4000 (GE Healthcare Life Sciences).