Pvdf membrane

Cells or exosomes were lysed using radioimmunoprecipitation assay buffer supplemented with 1-mM phenylmethylsulfonyl fluoride and phosphatase inhibitors (Beyotime, China) as described previously [27 (link)]. The concentrations of extracted proteins were determined using a BCA protein assay kit (Beyotime, China) according to the manufacturer’s instructions. The extracted proteins were added to 8% polyacrylamide gel and separated through electrophoresis. The separated proteins were transferred to PVDF membranes (Millipore, USA). Following the incubation of the PVDF membranes with 5% skimmed milk for 1 h at room temperature, the PVDF membranes were incubated with primary antibodies overnight at 4 °C. Then, the membranes were washed using TBST buffer and incubated with secondary antibodies for 1 h at room temperature. After washing the membrane with TBST buffer, the protein bands on the PVDF membranes were detected with enhanced chemiluminescence (ECL) reagent (Advansta, USA) under an infrared imaging system (Li-COR, USA). The primary and secondary antibodies used for western blotting are listed in Additional file 6: Table S2.

Tissues were homogenized as previously described and samples were loaded in 8% polyacrylamide gels, separated by SDS-page, and transferred to a PVDF membrane (Advansta, San Jose, CA, USA). Membranes were incubated with the specific primary antibodies overnight at 4 °C (listed below), and then incubated for 2 h at room temperature with secondary antibodies. The secondary antibodies were anti-mouse (GE Healthcare, Chicago, IL, USA, EUA) and anti-rabbit (Bio-Rad, Des Plaines, IL, USA). Membranes were revealed using ECL substrate in a Versadoc system (Bio-Rad, Des Plaines, IL, USA) and analyzed with Image Quant^® (Molecular Dynamics, Sunnyvale, CA, USA).

The Western blot analysis were performed in both cells (3T3-L1 cells treated with EA-Aa 31.25–500 µg.mL⁻¹ for 24 h) and organs (EAT, heart, kidney, liver and aorta). Cells and organ samples were washed with PBS and disrupted in lysis buffer (0.25 M Tris-HCl, 125 mM NaCl, 1% Triton-X-100, 0.5% SDS, 1 mM EDTA, 1 mM EGTA, 20 mM NaF, 2 mM Na₃VO₄, 10 mM β-glycerophosphate, 2.5 mM sodium pyrophosphate, 10 mM PMSF, 40 µL of protease inhibitor) using the TissueLyser systems (Quiagen, Germany). The BCA Protein Assay Kit was carried out on the supernatant of the centrifugation of samples (14.000 rpm for 20 min at 4 °C), followed by the addition of Laemmli buffer (62.5 mM Tris-HCl, 10% glycerol, 2% SDS, 5% β-mercaptoethanol, 0.01% bromophenol blue) [18 (link)]. Samples (20 μg) were loaded into SDS-PAGE and electroblotted onto PVDF membrane (Advansta, San Jose, CA, USA). Membranes were blocked with TBS-T 0.01% and BSA 5%, then incubated with the primary (overnight, 4 °C) and secondary antibodies (2 h, room temperature), following the dilutions suggested by the manufacturers. Immunoblots were detected with ECL substrate and the Versadoc system (Biorad, Hercules, CA, USA).

Hepatic and VAT samples were collected and washed with PBS and disrupted in lysis buffer (0.25 M Tris-HCl, 125 mM NaCl, 1% TritonX-100, 0.5% SDS, 1 mM EGTA, 1 mM EDTA, 20 mM NaF, 2 mM Na3VO4, 10 mM βglycerophosphate, 2.5 mM sodium pyrophosphate, 10 mM PMSF, 40 µL of protease inhibitor) using the TissueLyser system (Quiagen, Hilden, Germany). The bicinchoninic acid (BCA) Protein Assay Kit was carried out on the supernatant (14,000 rpm for 20 min at 4 °C, followed by the addition of Laemmeli buffer (62.5 mM Tris-HCl, 10% glycerol, 2% SDS, 5% β-mercaptoethanol, and 0.01% bromophenol blue). Tissue samples (20 µg) were loaded onto SDS-PAGE and electroblotted into polyvinylidene difluoride (PVDF) membrane (Advansta, San Jose, CA, USA). Tris-buffered saline-tween (TBS-T) 0.01% and bovine serum albumin (BSA) 5% were used to block the membranes, which were then incubated with primary (overnight, 4 °C) and secondary antibodies (2 h RT), following the dilutions listed in the Supplementary Table S1. The proteins of interest were detected using enhanced chemiluminescence (ECL) substrate with the LAS 500 system (GE Healthcare, Chicago, IL, USA). The bands of interest were quantified with Image Quant 5.0 software (Molecular Dynamics). The results were expressed as a percentage of control and normalized for the loading control (calnexin, 83 kDa).

Tissues were disrupted in lysis buffer (0.25 M Tris-HCl, 125 mM NaCl, 1% Triton-X-100, 0.5% SDS, 1 mM EDTA, 1 mM EGTA, 20 mM NaF, 2 mM Na₃VO₄, 10 mM β-glycerophosphate, 2.5 mM sodium pyrophosphate, 10 mM PMSF, 40 µL of protease inhibitor), centrifuged (14,000 rpm, 20 min, 4 °C) and denaturated with Laemmli buffer (62.5 mM Tris-HCl, 10% glycerol, 2% SDS, 5% β-mercaptoethanol, 0.01% bromophenol blue). Protein was quantified through the BCA Protein Assay Kit [34 (link)]. Samples were loaded in SDS-PAGE and electroblotted onto PVDF membrane (Advansta, San Jose, CA USA). For dot blot, 1 μL of denaturated samples were directly leaded to nitrocellulose membranes. All membranes were blocked with TBS-T 0.01% and BSA 5%, then incubated with the primary and respective secondary antibodies anti-mouse (GE Healthcare, Chicago, IL, USA), anti-rabbit and anti-goat (Bio-Rad Portugal, Lisboa, Portugal). Calnexin was used as loading control. Immunoblots were detected with ECL substrate and the Versadoc system (Bio-Rad).