Anti cnn1

Paraffin-embedded sections were firstly deparaffinized and then incubated with rabbit polyclonal anti-Ki-67 (#12075, Cell Signaling Technology) or rabbit polyclonal anti-CNN1 (#17819, Cell Signaling Technology) primary antibody at 4°C overnight. After being washed by TBST three times, the sections were then incubated with horseradish peroxidase–conjugated goat anti-mouse antibody (#4414, Cell Signaling Technology). The sections were washed by TBST three times and the signal was tested utilizing DAB Substrate Kit. Images were gained utilizing a microscopy.

Western blotting experiments were performed as previously described [30 (link)]. Briefly, total protein of VSMCs and vascular tissue was extracted with RIPA lysis buffer (Beyotime, Shanghai, China) and protease inhibitor (Thermo Fisher Scientific, CA, USA) mixture. The protein concentration was measured using a BCA kit (Thermo Fisher Scientific, CA, USA). Equal amounts of protein were separated by 12.5% SDS-PAGE and then transferred to PVDF membrane (MilliporeSigma, MA, USA), blocked in 5% milk for 1 h at room temperature. Then, they were incubated with the following primary antibodies at 4 °C overnight: anti-SLC6A6 (1:1000; Cat#ab236898; Abcam, MA, USA), anti-α-SMA (1:10,000; Cat#55135-1-AP; Proteintech, Wuhan, China), anti-SM22α (1:10,000; Cat# ab14106; Abcam), anti-SMMHC (1:1000; Cat#21404-1AP; Proteintech), anti-CNN1 (1:1000; Cat#17819; Cell Signaling Technology, MA, USA), anti-cyclin D1 (1:10000; Cat#ab134175; Abcam, MA, USA), anti-β-catenin (1:1000; Cat# ab32572; Abcam, MA, USA), and anti-β-actin (1:1000; Cat# 66009-1-Ig; Proteintech,Wuhan, China). The membranes were then incubated with anti-mouse or anti-rabbit HRP-conjugated antibodies (1:10,000; EasyBio, Beijing, China). Images were acquired using an optical scanner and analyzed using ImageJ software ( ImageJ software v1.6.0, MD, USA) [31 (link)].

Total protein of cells and vascular tissues was extracted with protein lysis buffer (Beyotime, China) and protease inhibitor mix (Thermo Fisher Scientific, USA). A BCA kit (Thermo Fisher Scientific, USA) was used for protein quantification. The protein extract denatured at 98°C was then separated by 10% SDS-PAGE, transferred to a 0.4 μm pore size PVDF membrane and blocked in a 5% skim milk solution for 1 h at room temperature. After washing in TBST solution, the membranes were incubated overnight at 4°C in the following antibody solutions: anti-α-SMA (1:10,000; Proteintech, Cat# 55135-1-AP, China), anti-SMMHC (1:1,000; Proteintech, Cat# 21404-1AP), anti-CNN1 (1:1,000; Cell Signaling Technology, Cat# 17819, USA), anti-PCNA (1:10,000; Abcam, Cat# ab92552, USA), anti-CBX3 (1:1,000; Proteintech, Cat #11650-2-AP), and anti-β-actin (1:1,000; Proteintech, Cat# 66009-1-Ig). On the following day, the membranes were washed and incubated in the corresponding secondary antibody solutions (1:10,000, EasyBio, China, Cat# BE0132-100) for 1 h at room temperature. An ECL Chemiluminescence Kit (Millipore, USA) was used to detect the protein signal. ImageJ was used for gray value analysis.