Myh11

Thoracic aorta were collected from DGCR8iKO or control mice and sonicated in lysis buffer (Thermo Scientific, Rockford, IL) containing 1% Halt proteinase inhibitor mixture (Thermo Scientific). Primary VSMCs isolated from DGCR8iKO and control mice were also collected to detect the expression of DGCR8 or VSMC marker gene expression. Approximately 80 µg protein for each sample was loaded on 8% SDS-PAGE gels and transferred to nitrocellulose membranes, which was then blocked with 5% nonfat milk for 1 h and incubated with primary antibodies against DGCR8, MYH11, Ki67, SM22 (Santa Cruz Biotechnology, Santa Cruz, CA), β-actin, SMA, and GAPDH (Sigma), phospho(p)-ERK1/2, phosphor (p)-AKT, ERK1/2, AKT (Cell Signaling Technology, Danvers, MA).

The proteins were extracted from the cells in a protein lysis buffer (Cell Signaling Technology, MA, USA) and subsequently centrifuged at 22,000 g for 5 min at 4°C to remove cellular debris. After boiling for 5 min, the lysate samples were separated by a 12% SDS-PAGE electrophoresis and electrotransferred to a PVDF membrane. The membrane was blocked in TBST containing 5% BSA and incubated with anti-P2X7, P2Y1, P2Y2, P2Y11, VEGFR2, VE-cadherin, PECAM-1, calponin, SMA-α, MYH-11 (1 : 500), or GAPDH antibodies (1 : 5,000) (Santa Cruz Biotechnology, CA, USA) overnight at 4°C. The membranes were washed three times with TBST and incubated with the secondary antibodies (1 : 5,000) (CALBIOCHEM, CA, USA) for 60 min at RT. After washing with TBST, immune-detection was accomplished by using the Luminata Forte Western HRP substrate (Merck Millipore, MA, USA) and images were taken using Bio-Rad Chemidoc system.

Aortic proteins were extracted as previously described [22 (link)]. The protein concentration of aortic proteins and SMC cell extracts was standardized with a Bio-Rad protein assay. Equal amounts (25 μg) of aortic tissue extracts or aortic SMC extract from WT and mgR mice were loaded under reducing conditions onto a 10% SDS-polyacrylamide gel and transferred to a polyvinylidene difluoride (PVDF) membrane (Amersham Biosciences) The membranes were then incubated with the following primary antibodies: TGF-β (Cell Signaling 3711S), KLF4 (Cell Signaling 4038S), α-actin (Santa Cruz Biotechnology, (1A4) sc-32251), Tropoelastin (Elastin Products Co. PR385), MYH11 (Santa Cruz Biotechnology, (H-44) sc-98705, Calponin (Santa Cruz Biotechnology, (FL-297) sc-28545), SM22α (Santa Cruz Biotechnology (H-75) sc-50446), β –actin (Cell Signaling 4967L) and GAPDH (Cell Signaling 5174S). The bound primary antibody was detected with HRP-linked anti-mouse or anti-rabbit IgG (Cell Signaling 7076S and 7074S). Immunoreactive bands were visualized by autoradiography using ECL (Amersham Biosciences). Gelatin zymography for aortic tissue extract and SMC conditioned media was performed as described previously by Longo et al. [23 (link)], with 0.8% gelatin in a 10% SDS-polyacrylamide gel. The molecular sizes were determined using protein standards (Fermentas).