Beclin 1 antibody

Reagents used were CA125 IRMA kit (Tianjin Xiehe Medical Science & Technology Co., Ltd., cat. no. RC70417); E₂, P, FSH, and LH ELISA kits (Cayman Chemical, Ann Arbor, MI, USA, cat. no. 501890, 582601, 500710, and 500720); TLR4 antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA, cat. no. Sc-293072); NF-κB/P65 antibody (Abcam, Cambridge, UK, cat. no. AB16502); Beclin-1 antibody (ProteinTech, Wuhan, China, cat. no. 11306-1-AP); SQSTM1/P62 antibody (Bioss, Beijing, China, cat. no. Bs-55207r); GAPDH (Servicebio, Wuhan, China, cat. no. P04406); and HRP-labeled Goat Anti-Rabbit IgG (Servicebio, Wuhan, China, cat. no. GB23303).

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.