Sm mhc

Western blot test was used to identify expression of SMC proteins. Cells were lysed in RIPA buffer with protease and phenylmethylsulfonyl fluoride (PMSF; Roche, Indianapolis, IN, USA). The protein concentration was assayed using the BCA method (Bio-Rad). Approximately 20 to 50 μg of total protein samples was loaded on a 10% SDS-PAGE after denaturation by boiling for 10 min. The separated proteins transferred to polyvinylidene difluoride membranes. Membranes were blocked by incubation in Tris-buffered saline containing 0.05% Tween 20 and 5% skimmed milk with constant shaking for 1 h. The membrane was probed with antibodies against a-SMA (1 : 1000, Abcam, Cambridge, UK), calponin (1 : 1000, Abcam, Cambridge, UK), SM22a (1 : 1000, Abcam, Cambridge, UK), SM-MHC (1 : 1000, Abcam, Cambridge, UK), smoothelin-like 2 (1 : 2000, Santa Cruz, Dallas, USA), ACLP (1 : 1000, Abcam, Cambridge, UK), CaMKIIα (1 : 5000, Abcam, Cambridge, UK), CaMKIIβ (1 : 1000, Abcam, Cambridge, UK), CaMKIIγ (1 : 500, Abcam, Cambridge, UK), and CaMKIIδ (1 : 1000, Santa Cruz, Dallas, USA) overnight at 4°C. GAPDH was used as an internal loading control. The membranes were washed three times with TBST and incubated with the appropriate secondary antibodies at room temperature for 1 h and detected using enhanced chemiluminescence.

The membrane protein and cytoplasm protein were isolated from VSMCs through membrane and cytosol protein extraction kit (Beyotime, China). The proteins were quantified using BCA kit from Vazyme (China), mixed with 5x SDS loading buffer and electrophoresed on a 10% SDS-PAGE gel. The anti-CD137 polyclone antibody (Abcam, USA) was used to detect the monomer or polymer of CD137. Antibodies such as SM-MHC (Abcam, USA), NFATc1 (CST, USA), vimentin (Immunoway, USA), calponin (Abcam, USA), and α-SMA (Sigma, USA) were used to observe the phenotype transformation of CD137 axis.

The antibodies used for the current study are listed below. KDR/VEFGR2 (mouse monoclonal; ab9530; Abcam); α-SMA (mouse monoclonal; A5228; Sigma); troponin T (mouse monoclonal; clone 13–11; Thermo Fisher); α-sarcomeric actinin (mouse monoclonal; A7811; Sigma); FLAG tag (mouse monoclonal; F-tag-01; Applied Biological Materials); Thy1/CD90 (Mouse monoclonal; clone 5E10; BD Pharmingen); SM-MHC (rabbit polyclonal; Abcam); ANP (mouse monoclonal; clone 23/1; Santa Cruz); BNP (mouse monoclonal; clone 50E1; Thermo Fisher); c-kit (rabbit monoclonal; clone YR145; Epitomics); α-tubulin (mouse monoclonal; clone DM1A; Sigma); V5 tag (mouse monoclonal; clone E10; Applied Biological Materials). For staining, the antibodies were diluted 1:100–1:250, and for Western blots, antibodies were diluted 1:500–1:1000 as recommended by the manufacturers.

The collected penile specimens were lysed in RIPA buffer (Cell Signaling Technology, USA) containing a protease inhibitor cocktail (KeyGEN BioTECH, China). The proteins were separated by SDS-PAGE and transferred to a PVDF membrane (Millipore, USA). After blocking, the membrane was incubated with primary antibodies against desmin (Abcam, USA), SM-MHC (Abcam, USA), Collagen1 (Abcam, USA), MMP2 (Abcam, USA), p-SMAD2 (Abcam, USA), SMAD2 (Abcam, USA), and β-actin (Abcam, USA). After washing the membrane three times, appropriate secondary antibodies were applied and incubated with the membrane for 1 h. Protein bands were detected by ECL chemiluminescence reagents (KeyGEN BioTECH, China) and imaged with an imaging system (UVP GDS-8000, USA).

Cerebral arteries and A7r5 cells lysis buffer were prepared in M‐PER Mammalian Protein Extraction Reagent (Thermo) with freshly 1% protease inhibitor cocktail (Thermo). After centrifugation, supernatants were denatured for Western blotting. Proteins were separated using NuPAGE 4%–12% Bis‐Tris gel (Invitrogen), and then transferred to polyvinylidene fluoride (PVDF) membranes (Millipore). Membranes were blocked and subsequently incubated with appropriate primary antibodies against SM‐MHC (Abcam), SM‐α‐actin (Abcam), SM22α (Proteintech), OPN (Abcam), PCNA (Abcam), Ca_V3.1 (Alomone labs), Ca_V3.2 (Affinity), NFATc1‐4 (Bioss), glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH) (Proteintech) and Histone‐H3 (Abcam) at 4°C overnight and horseradish peroxidase (HRP)‐conjugated secondary antibodies (Abcam). Proteins were detected and visualized using the chemiluminescent HRP substrate (Millipore). Image J software was applied for quantification.

The male Sprague-Dawley rat primary cerebral VSMCs of basilar arteries and circle of Willis arteries (ICell Bioscience, China) were cultured in dulbecco modified eagle medium and ham’s F-12 medium (DMEM/DF12, Gibco, USA, 1:1) supplemented with 10% fetal bovine serum (FBS, Gibco, USA) in 5% CO₂ at 37 °C, and medium was replaced every 48 hours. Cells were confirmed by α-SMA (Abcam, United Kingdom, 1:200) and SM-MHC (Abcam, United Kingdom, 1:200) immunostaining. The cells with 85% confluence were subcultured. VSMCs less than 6 passages with 65% of confluence were used in the following experiments. To mimic SAH pathophysiology procedure and optimal stimulating concentration, cells were exposed to hemoglobin (Sigma, USA) at different concentrations ranged from 1 μM to 20 μM for 24 hours before following assays. After hemoglobin administration, cellular morphology was observed by inverted phase contrast microscope (Olympus, Japan) regularly, and western blots were used to detect the expression of α-SMA and Smemb in different concentrations groups.