P smad2

Histology of the aortic root and the mid-ascending aorta for LDLR^-/-Apobec1^-/- mice was performed 21 days after MI or sham procedure. Blood was removed from the arterial system by infusion of isothermic buffered saline after which the aorta was perfusion-fixed. Trans-axial sections of the aortic root at the level of the sinuses were stained with Masson’s trichrome to assess the plaque area within the internal elastic lamina, and collagen content. Immunofluorescence histology was performed with anti-mouse primary mAb against Mac-2 (M3/38, Invitrogen, Waltham, Massachusetts, USA) for monocytes/macrophages, against CD41 (ab181582, Abcam, Cambridge, United Kingdom) for platelets, and against matrix metalloproteinase-9 (MMP-9) (PA5-13199, ThermoFisher). Staining for evidence of vascular cell signaling through platelet-derived TGFβ1 was performed with mAb against β-catenin (51067-2-AP, Proteintech, Rosemount, IL) and phosphorylated SMAD2 (pSMAD2) (H.205.4, Invitrogen). Secondary staining was performed with species-appropriate secondary polyclonal antibodies labeled with ALEXA fluorochromes (Fluor-488, Fluor-568, or Fluor-647) and observations were made by confocal fluorescent microscopy (TCS SP5, Leica Microsystems, Buffalo Grove, IL). Spatial extent of plaque size, collagen content, or fluorescent staining area were quantified using Image-J.

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.

Cells or a 3-cm length section of injured spinal cord was lysed in lysis buffer on ice. Following electrophoresis, the collected proteins were transferred to a PVDF membrane and incubated overnight with primary antibodies at 4 °C (Cnpase, 1:500, Sigma, Germany; BMP2, 1:1000, Sigma, Germany; TGF-β, 1:1000, Invitrogen, USA; p-Smad2, 1:1000, Invitrogen, USA). The membrane was then incubated 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 chemiluminescence reagent (Thermo Scientific) and quantified using ImageJ software. The full-length original gels are included in Additional file 2: Figure S2.