Clarity max substrate

Cells were re-suspended in NuPAGE sample buffer supplemented with 50 mM of dithiothreitol (DTT) and loaded on 4–12% NuPAGE Bis-Tris Protein Gels and electrophoresed at 180 V for 1 h. Gels were transferred to Nitrocellulose membranes using wet transfer system XCell II Blot Module (Invitrogen) at a constant amperage of 350 mA for 2 h. Membranes were blocked with 5% skimmed milk in Tris Buffered saline-Tween for 1 h before incubation with primary antibody anti-myc tag monoclonal clone 4A6 (Millipore) diluted to a final concentration of 200 ng mL⁻¹ in 3% skimmed milk in TBST for 1 h at room temperature. After washing membranes proceeded to incubation with secondary antibody anti-mouse IgG (H + L) conjugated to HRP (Molecular Probes) at 300 ng mL⁻¹ in 3% skimmed milk in TBST for 1 h at room temperature. Membranes were washed, incubated with Clarity Max substrate (BioRad) and developed in ChemiDoc MP (BioRad).

OXTR protein expression was examined in adipose tissue membrane fractions. Adipose tissue was homogenized in ice-cold lysis buffer (15 mM KCl, 1.5 mM MgCl₂, 10 mM HEPES, 1 mM DTT, protease inhibitors), centrifuged at 1000 x g for 5 min at 4 °C, and OXTR enriched membrane fractions in the supernatant were isolated by centrifugation at 100,000 g × 60 min at 4 °C. The membrane pellet was solubilized in SDS lysis buffer (50 mM Tris, pH 8.6, 1% SDS). Protein was measured with BCA Protein Assay (Pierce, Rockford, IL, USA). OXTR protein expression was evaluated using previously described methods and using a specific polyclonal anti-rabbit OXTR (cat. no. ab181077; Abcam, Cambridge, MA, USA) validated against tissues from OXTR knockout mice [10 (link)]. Immunoreactive bands were detected with appropriate peroxidase conjugated secondary antibody for 1 h and then visualized with chemiluminescence (Clarity Max substrate, Bio-Rad Laboratories, Hercules, CA, USA). To normalize for protein loading, the membranes were stripped and re-probed with chicken anti-actin antibody (Cat. no. SAB3500350; Sigma, St. Louis, MO) or sodium/potassium ATPase (Cat. no. ab76020; Abcam, Cambridge, MA, USA).

Protein concentration was measured by the Bicinchoninic acid (BCA) assay. EV lysates (approximately 3 µg per sample) and cell lysates (approximately 10 µg per sample) were mixed with Laemmli buffer and heated at 95 °C. Samples were run on a 4–20% Tris/glycine gel (Invitrogen, Carlsbad, CA, USA) and then transferred to a PVDF membrane overnight. Blocked membranes were incubated with the appropriate primary antibody overnight at 4 °C. The primary antibodies used included α-CD63, α-CD81, α-CD9, α-GAPDH, α-Caspase 3, α-Cyclin A, α-Cyclin D1, α-Cyclin E, α-N-cadherin, α-E-cadherin, α-CD142, α-VEGF, α-DNA Polymerase β, α-p-AKT, and α-Akt, and α-Actin. See Table 1 below for primary antibody catalog information. After incubation with the appropriate secondary antibody, membranes were developed with Clarity™ or Clarity Max™ Substrate (Bio-Rad, Hercules, CA, USA), and images were taken with the ChemiDoc™ Imaging System (Bio-Rad). Images of full-length blots are included in Figure S1.