Anti rock1

After the cell migration model was removed and the cells were incubated for 72 h, the BMSCs from the four types of PET-AL sheets were harvested using pancreatic enzyme and rinsed three times with PBS. RIPA buffer was added to lyse the cells. The lysates were centrifuged at 10⁴ ×g for 10 min at 4°C, and the protein in the supernatant was extracted. Equal amounts of protein (30 μg) were separated by 10% SDS-PAGE and transferred to a PVDF membrane (poly-vinylidene fluoride, Bio-Rad). After blocking with 5% nonfat dry milk in Tris-buffered saline with Tween for 1 h, the membranes were incubated with the following antibodies overnight at 4°C: anti-RhoA (Santa Cruz Biotechnology, Inc., USA), anti-ROCK1 (Santa Cruz Biotechnology, Inc., USA), anti-pMLC (Cell Signaling Technology, Inc., USA), and anti-GAPDH (Santa Cruz Biotechnology, Inc., USA), followed by incubation with horseradish peroxidase-conjugated antibody for 1 h at room temperature. Signals were detected using the Western-Light Chemiluminescent Detection System (Peiqing, China). The integrated density values were calculated using Image Pro Plus 6.0.

Cells were lysed in JS buffer (50 mM HEPES, pH 7.5, containing 150 mM NaCl, 1% glycerol, 1% Triton X-100, 1.5 mM MgCl₂, 5 mM EGTA, 1 mM Na₃VO₄, and 1X protease inhibitor cocktail). Protein concentration was determined with the Bradford assay (BioRad, Milan, Italy) using bovine serum albumin as the standard, and equal amounts of proteins were analyzed by SDS-PAGE (12% acrylamide). Gels were electroblotted into nitrocellulose membranes (G & E Healthcare, Milan, Italy). Membranes were blocked for 1 h with 5% non-fat dry milk in tris-buffered saline (TBS) containing 0.1% Tween-20, and incubated at 4°C overnight with the primary antibody. Detection was performed by peroxidase-conjugated secondary antibodies, using the enhanced chemiluminescence system (Thermo Euroclone, Milan, Italy). Primary antibodies used were: anti-NRAS, anti-ERK1, anti-SKP2, anti-ROCK1 (Santa Cruz Biotechnologies, MA, USA), anti-pP42/44, anti-pAKT, anti-AKT, anti-pmTOR, anti-mTOR, anti-pGSK3β, anti-GSK3β, anti-pELK, anti-ELK (Cell Signaling, Danvers, MA, USA), and anti-βactin (Sigma Aldrich, Milan, Italy).

Total protein was extracted from artery rings, and the protein concentration determined using a bicinchoninic acid (BCA) protein assay kit (Thermo Fisher Scientific, Waltham, MA, USA). Immunoblotting was performed with the following primary antibodies: anti-RhoA (Bioworld), anti-ROCK1 (Santa Cruz Biotechnology), anti-ROCK2 (Santa Cruz Biotechnology), anti- K_v1.2, or anti-K_v1.5. HRP-conjugated anti-rabbit secondary antibody was used at a dilution of 1:2000. Detection of GAPDH (diluted 1:1500; #5471; Cell Signaling Technology, Danvers, MA, USA) served as an internal loading control. All blots were scanned with the LabWorks image processing system (UVP Inc., Upland, CA, USA). Protein band pixel values were calculated using Gel-pro Analyzer 4.0 (Media Cybernetics Inc., Rockville, MD, USA).