Nf κb antibody

We included 9 ESRD patients, GG (n = 3), GT (n = 3), and TT (n = 3), for the ChIP assay. ChIP assays were conducted by using the ChIP Kit ab500 (Abcam, USA) and NF-κB antibody (Proteintech) according to the manufacturer’s instructions. The immunoprecipitate was eluted with 100 μl DNA purifying slurry, and 2 μl of DNA was used in qPCR. Input DNA and NF-κB-enriched DNA fragments were amplified by using qPCR in a 7500 Fast Real-Time PCR System (Applied Biosystems) with primers 5′-ATTCTCACCATGGGAATGG-3′ and 5’GAGGACAGCAAGGTAATAG-3′. The results are shown as percentage input.

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.

Human adiponectin (Acrp30) and human TGF-β1 were purchased from Novoprotein (Beijing, China). Cell Counting Kit-8 (CCK-8) and EdU kits were procured from SolarBio (Beijing Solebo Technology Co., Ltd, Beijing, China). PCNA antibody, smooth muscle actin polyclonal (α-SMA) antibody, collagen type I (collagen I) rabbit polyclonal antibody, p38 MAPK antibody, p-p38 MAPK antibody, NF-κB antibody, p-NF-κB antibody, β-catenin antibody, p-β-catenin antibody, Notch1 antibody, and HRP-conjugated AffiniPure goat anti-mouse IgG (H + L) were obtained from Proteintech (Wuhan, China). SB203580 was purchased from MCE (Shanghai, China). Other chemicals used in this study, such as the BCA protein assay kit and SDS-PAGE gel separation kit, were acquired from ASPEN (ASPEN Biotechnology Co., Ltd, Wuhan, China).