Lc3 antibody

Each sample consisted of two mesenteric arteries from the same group. RIPA lysis buffer containing proteinase inhibitors was added, and homogenate was prepared. A BCA kit was used for protein quantification. The extracted protein was subjected to SDS-PAGE followed by isolation and PVDF membrane transfer. Evaporated milk (5%; prepared with T-TBS buffer) was applied for sample blocking. The primary antibodies included ET_A receptor antibody (dilution, 1 : 500; GeneTex, Inc., USA), Class II PI3K antibody (1 : 1000; Cell Signaling Technology), p-NF-κB p65 antibody (1 : 1000; Cell Signaling Technology), Beclin-1 antibody (1 : 3000; Proteintech Biotechnology, Inc., USA), LC3 antibody (1 : 500; Proteintech Biotechnology), p62 antibody (1 : 3000; Proteintech Biotechnology), NF-κB antibody (1 : 3000; Proteintech Biotechnology), and GAPDH antibody (1 : 1000; Proteintech Biotechnology), and they were prepared with PBS containing 2% bovine serum albumin as the diluent. The secondary antibodies were peroxidase-labeled goat anti-rabbit IgG and goat anti-mouse IgG (both, 1 : 5000). Images were taken with Image Gauge Ver. 4.0 (Fuji Photo Film Co., Ltd, Japan) for optical density analysis. Grayscale self-correction was performed. The level of the target protein is presented as the ratio between the band grayscale of the target gene and that of the housekeeping gene.

MTT, acridine orange, di-2-pyridylketone, pifithrin-α, and monodansylcadaverine (MDC) were purchased from Sigma-Aldrich. LC3 antibody was obtained from Proteintech Group (Wuhan, China), caspase-8, GAPDH, bax, cytochrome c, and bcl-2 were purchased from Boster (Wuhan, China). RPMI 1640 and fetal bovine serum were purchased from Zhejiang Tianhang Biological Technology Co. Ltd.

MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide), ethidium bromide (EB), di-2-pyridylketone, RPMI-1640, and other chemicals were purchased from Sigma-Aldrich (Shanghai, China). LC3 antibody was obtained from Proteintech Group (Wuhan, China). Antibodies against cyclin D1, caspase 3, β-actin, Bax, and Bcl-2 were purchased from Boster (Wuhan, China).

Tissue slides were prepared in a smiliar manner to those used in H&E staining. After antigen repair, goat serum was inoculated at room temperature for 30 min, the CD31 antibody (ab182981; Abcam) was incubated overnight, and a secondary antibody solution of Goat Anti-Rabbit IgG (HRP) (ab205718; Abcam) was added. Antigen repair was performed after the first dyeing and cleaning. Donkey serum was inoculated at room temperature for 30 min, the LC3 antibody (1:200; Proteintech, USA) was incubated overnight, and Donkey Anti-Rabbit 594 secondary antibody (Abcam, ab150075) was added through a drip. We used 4′, 6-Diamidino-2-phenylindole (DAPI, #C0060; Solarbio) to stain the cell nuclei and images were captured using laser confocal microscopy (Nikon, Tokyo, Japan).