Smad7

Western blot analysis of kidney tissue homogenates was performed to analyze SNAIL1, TWIST1, SMAD3, and SMAD7 protein expression (Invitrogen, Waltham, MA, USA). According to our previous real-time PCR analysis, GAPDH (Invitrogen, Waltham, MA, USA) was also used as a housekeeping protein. Proteins were extracted from kidney tissue using P-TER solution (Thermo Fisher Scientific, USA) and quantified by the BCA protein assay kit (Thermo Fisher Scientific, USA). A quantity of 50 µg of total proteins were separated by 10% polyacrylamide gel electrophoresis (SDS-PAGE) under reducing conditions and transferred to nitrocellulose membranes (Thermo Fisher Scientific, Waltham, MA, USA). Blocking was performed using 5% non-fat dry milk in 1X TBST for 1h at 4 °C with constant agitation. Membranes were incubated overnight at 4 °C with primary antibodies diluted 1:1000 (SNAIL1, TWIST1, SMAD3, and GAPDH) and 1:500 (SMAD7) in 1X TBST. Antibody binding was revealed with an HRP-conjugated secondary anti-antibody diluted 1:5000 in 1X TBST using a BM Chemiluminescence kit (Roche Diagnostics, Indianapolis Ind, Indianapolis, IN, USA). Densitometric analysis was performed with a UVP ChemiStudio image analyzer (Analytik Jena, Jena, Germany) using the VisionWorks^® software (Analytik Jena, Germany). The semiquantitative analysis of every protein was shown as normalized levels.

Heart tissue lysates were prepared by homogenization in lysis buffer (50 mM Tris-HCl, 150 mM NaCl, 0.25% Triton X-100, pH 7.4) supplemented with a protease inhibitor cocktail (Boehringer Mannheim). Approximately 50 μg of protein from each sample was separated by SDS-PAGE and transferred to a PVDF membranes (Schleicher & Schuell). The membranes were blocked with 5% non-fat milk and then incubated with primary antibodies at 4°C overnight. Antibodies for TGF-β2 and CD31 were purchased from Abcam, SMAD7 from Invitrogen, vimentin from Santa Cruz Biotechnology, Cytl1, HA, and α-SMA from Sigma-Aldrich, GAPDH, phospho-SMAD2, SMAD2/3, and VE-cadherin from Cell Signaling. The membranes were incubated with secondary antibodies conjugated to horseradish peroxidase (Jackson ImmunoResearch) and then developed with a chemiluminescent substrate (Perkin Elmer).

Cells, EVs or a 0.5 cm length section of injured spinal cord (centered on the epicenter of the injured lesion) were lysed in lysis buffer on ice. Ten micrograms of collected proteins were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS‒PAGE) and transferred to a PVDF membrane. The membrane was then incubated with primary antibodies at 4 °C overnight (SMAD 7, 1:1000; p-Smad2, 1:1000, Invitrogen, USA) and with the secondary antibodies (Elabscience; 1:5000 in blocking solution) at room temperature for 1 h. The blots were then visualized using the SuperSignal West Pico enhanced chemoluminescence reagent (Advansta, USA) and quantified using ImageJ.