Enhanced chemiluminescence prime western blotting detection reagent

To determine if MVs affect MAPK signaling, recipient VSMCs were incubated with and without 10 μg MVs for 30 minutes, 2 hours, and 4 hours, at 37°C; total protein from the co-culture was isolated using lysis buffer as previously described.^{54 (link)} The activation of MAPK was assessed using a PathScan MAP Kinase Multi-Target Sandwich ELISA Kit. To confirm the MAPK signaling by cellular and media MVs, Western blot analyses were performed. Briefly, 20 μg of protein was loaded on 10% SDS-PAGE, and the blots were incubated with antibody against Phospho-MEK1 or Phospho-Erk1/2 (1:500; Cell Signaling Technology, Danvers, MA) overnight, at 4°C, followed by incubation, with peroxidase-conjugated secondary antibody (1:5000 dilution), and immunodetection, with the Enhanced Chemiluminescence Prime Western Blotting Detection Reagent (Amersham Biosciences, Piscataway, NJ). For loading control, western blotting analysis was also performed using antibodies against total MEK1 or total Erk1/2 (1:100; Cell Signaling Technology, Danvers, MA). The band intensity was analyzed using a ChemiDoc MP Imaging System (Imaging Lab 4.0), and MAPK activation was quantified by normalizing phosphorylated MAPK to total MAPK.