Pecam1

Cell lysis was performed with RIPA lysis buffer (150 mM TrisHCL, 150 mM NaCl, 0.5% Deoxycholate, 0.1% SDS, 1% NP-40) for 30 min at 4 °C followed by centrifugation at 20,000xg for 10 min. Cell lysates were submitted to 6%- or 12% SDS-PAGE gels. Proteins were transferred to PVDF membrane. Inmunodetections were done with following primary antibodies: anti-smooth muscle myosin heavy chain 11 (ab82541, Abcam, Cambridge, UK) and anti-CD31 antibody (PECAM1-EPR3094 ProteinTech, Chicago, USA). Proteins were detected with Immobilon™ Western Chemiluminescence HRP Substrate detection reagent (Millipore, Billerica, MA, USA) and were visualized with the ChemiDoc™XRS Imaging System (Bio-Rad, Richmond, CA, USA). Anti-ACTIN antibody (A2066, Sigma-Aldrich, St Louis, MO, USA) was used as housekeeping for normalization. Data analysis was done using Image Lab™ Software (Bio-Rad, Richmond, CA, USA).

Norepinephrine bitartrate monohydrate was obtained from MedChem Express (Monmouth Junction, NJ, USA). Propranolol hydrochloride and losartan were bought from Tocris Bioscience (Bristol, Avon, UK). Phentolamine hydrochloride and GW4869 were purchased from Sigma (St. Louis, MO, USA). GW4869 was dissolved in 1% dimethyl sulfoxide (DMSO). PBS containing 1% DMSO was used as the control. Other chemicals were dissolved in PBS.
Antibodies against α-SMA (1:5000), CD9 (1:1000), TSG101 (1:2000), calnexin (1:2000) and ACE (1:1000) were purchased from Abcam (Cambridge, MA, USA). Antibodies against PCNA (1:5000) and PECAM-1 (1:2000) were obtained from Protein Tech Group Inc. Antibody against CD63 (1:1000) was acquired from Wanleibio. (Shengyang, China). Antibody against vimentin (1:2000) was purchased from Abways Technology, Inc. (Shanghai, China). Antibodies against β-actin (1:10000) and GAPDH (1:10000) were bought from Cell Signaling Technology (Beverly, MA, USA). These antibodies were used for Western blot analyses. GAPDH and β-actin were used as loading control.

Exogenous peroxidase was quenched using 3% H₂O₂ (MilliporeSigma), and 1% bovine serum albumin in PBS was applied. The sections were incubated overnight at 4°C with primary antibody, followed by anti-rabbit IgG (biotinylated) (1:100; Dako, Santa Clara, CA, USA). To visualize the immune complex, Extravidin Peroxidase (MilliporeSigma) was applied, followed by incubation with 3-amino-9-ethylcarbazole in acetate buffer with 0.015% H₂O₂, resulting in red/brown staining. Sections were counterstained with Alcian Blue and light green and mounted with hydro-mount. Primary antibodies (rabbit polyclonal) were obtained as follows: type I collagen (COL I, LF68, 1:1000) (kindly donated by Dr. Larry Fisher, National Institutes of Health, Bethesda, MD, USA), CD31 (PECAM-1, 1:100; Proteintech, Manchester, United Kingdom), type II collagen (COL-II, 1:500; Calbiochem, Merck-Millipore, Watford, United Kingdom), and von Willebrand factor (vWF) (1:100; DakoCytomation, Cambridgeshire, United Kingdom).