Anti slc6a6

For cellular immunofluorescence, VSMCs were fixed with pre-cooled methanol for 5 min, blocked with 10% goat serum, and incubated with the following primary antibodies at 4 °C overnight: anti-SLC6A6 (1:50; Abcam, MA, USA) and anti-α-SMA (1:100; Proteintech, Wuhan, China). Paraffin sections were subjected to routine dewaxing and antigen retrieval. The sections were blocked in 10% goat serum at room temperature for 1 h and incubated with the following primary antibodies at 4 °C overnight: anti-SLC6A6 (1:100; Abcam, MA, USA), anti-α-SMA (1:200; Proteintech, Wuhan, China), anti-cyclin D1 (1:50; Abcam, MA, USA), and anti-PCNA (1:100; Proteintech, Wuhan, China). Immunohistochemical staining was performed with a DAB kit (ZSGB-BIO, Beijing, China) according to the instructions. Images were obtained using a digital slice scanner (KFBIO, Ningbo, China). For immunofluorescence staining, cells or vascular samples were incubated with the fluorescent secondary antibodies Alexa Flour 488-conjugated goat anti-mouse (1:200, Proteintech, Wuhan, China) and Alexa Flour 594-conjugated goat anti-rabbit IgG (1:200, Proteintech, Wuhan, China) for 1 h at room temperature. Nuclei were stained with DAPI (Solarbio, Beijing, China). Fluorescent images were obtained using a confocal laser microscope (Nikon, A1R, Tokyo, Japan) [32 (link)].

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)].

Cells were washed twice with ice-cold PBS and treated with RIPA lysis buffer. Protein concentrations were quantified by the BCA protein assay kit (Beyotime, Haimen, China). Protein were separated on 8% SDS-PAGE gels, transferred onto PVDF membranes (Bio-Rad, Hercules, CA, USA) and blocked for 1 h at room temperature. Membranes were probed with primary antibodies anti-SLC6A6 (1:1000 dilution; Abcam, USA) at 4 °C overnight followed by incubation with HRP-conjugated secondary antibodies. Each sample was also treated with anti-β-actin antibody (Sigma-Aldrich) as a control. Blots were detected using an ECL detection system.