Immobilon western chemiluminescence hrp substrate detection reagent

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).

Protein extraction was carried out 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 20,000× g for 10 min in trypsinized VSMCs. Cell lysates were quantified by Pierce™ BCA Protein Assay Kit (23225, Thermo Fisher Scientific, Waltham, MA, USA) and 12 µg protein were loaded into 6% or 12% SDS-PAGE gels with reducing loading buffer. Then, proteins were transferred from SDS-PAGE gel to 0.45 µm-pore PVDF membrane. PVDF membranes were blocked with 2% casein solution following incubation with primary antibody anti-smooth muscle Myosin heavy chain 11 (ab82541, Abcam, Cambridge, UK) and then washed with TBS-Tween. The secondary antibody was anti-rabbit horseradish peroxidase conjugate (#7074, Cell Signaling Technology, MA, USA) and as housekeeping anti-GAPDH antibody (MAB374, Millipore, Billerica, MA, USA) was used for normalization. Proteins were detected with Immobilon™ Western Chemiluminescence HRP Substrate detection reagent (WBKLS0500, Millipore, Billerica, MA, USA) and were visualized with the ChemiDoc™ XRS Imaging System (Bio-Rad, Hercules, CA, USA). Data analysis was done by densitometry comparing MIT (n = 7) and PLQ (n = 7) cells and additionally, between A (n = 6) and S (n = 5) plaque VSMCs, both using Image Lab™ Software (Bio-Rad, Hercules, CA, USA).