Horseradish peroxidase conjugated anti rabbit secondary antibody

Western blotting was performed to evaluate the exosome markers. Briefly, the exosomal pellet was resuspended in 200 μL of RIPA buffer containing a phosphatase inhibitor cocktail, and the lysates were incubated at room temperature for 5 min after vortexing for 15 s. The concentrations were determined using a BCA assay kit (Invitrogen). The samples were boiled at 95 °C for approximately 5 min, and then chilled on ice for 5 min before being loaded onto the gel. The proteins were separated using a 12% sodium dodecyl sulfate-polyacrylamide gel and then transferred onto a polyvinylidene fluoride membrane (Bio-Rad Laboratories, Redmond, WA, USA). After blocking with 5% skim milk (BD Pharmingen, San Diego, CA, USA) for 1 h, the blots were incubated with indicated primary antibodies at 4 °C (exosome panel, Abcam #ab275018, Cambridge, UK), according to the manufacturer’s protocols. Subsequently, the blots were incubated with horseradish peroxidase-conjugated anti-rabbit secondary antibody (1:1000; GeneTex, Irvine, CA, USA) for 1 h at room temperature. The membrane was visualized using SuperSignal West Femto Maximum Sensitivity Substrate (Invitrogen), according to the manufacturer’s instructions. Finally, signals were detected using the LAS4000 system (GE Healthcare, Uppsala, Sweden).

Immunoblotting was performed according to the method described by Towbin et al. with modifications as previously described.²⁴, ²⁵ Briefly, cell lysates were electrophoresed on sodium dodecyl sulfate‐polyacrylamide gels and electroblotted onto polyvinylidene difluoride membranes (Immobilon‐P Transfer Membrane; Millipore). The membranes were blocked with Block Ace (blocking milk; Yukijirushi) and incubated with rabbit anti‐adiponectin antibody (cat. no. GTX107737, GeneTex Inc.) and anti‐T‐cadherin antibody.²⁶ For immunodetection, horseradish peroxidase‐conjugated anti‐rabbit secondary antibody (1:2000; cat. no. 7074); Cell Signaling Technology Inc. and ultrasensitive HRP substrate (TaKaRa Bio) were used. Images were obtained using an Invitrogen iBright 1500 gel imaging system (Thermo Fisher Scientific). After stripping immunoreactivity, the membranes were incubated with an anti‐GAPDH antibody (Sigma) to evaluate the input proteins.