Horseradish peroxidase labeled goat anti rabbit igg

A total of 150 mg of leaf sample was crushed in liquid nitrogen and incubated with extraction buffer (200 mM Tris‐Cl pH 8.0, 100 mM NaCl, 400 mM Sucrose, 10 mM EDTA, 100 mM PMSF, 0.05% Tween‐20, 28.6 mM β‐mercaptoethanol, 1X Protease Inhibitor Cocktail). Proteins were allowed to extract for 30 min at 4°C with gentle shaking. Samples were centrifuged at 13,000 rpm, 4°C, and supernatant was collected. Protein quantity was estimated with Bradford using BSA standards. A total of 40 μg of protein was electrophoresed on 12% SDS‐PAGE. Thereafter, proteins were blotted on to a polyvinylidene difluoride membrane (Hybond‐PVDF, Amersham, UK) according to the manufacturer's protocol. Membrane was blocked with 5% (w/v) skimmed milk in PBST buffer for 2 h at room temperature. Membrane was thereafter incubated with rabbit anti‐β‐glucuronidase (Invitrogen) (1:1,000) for 2 h followed by three gentle washing with PBST for 5, 10, and 5 min. Subsequently, membranes were incubated with secondary antibody (horseradish peroxidase‐labeled goat anti‐rabbit IgG) (Sigma‐Aldrich, USA) in 1:10,000 dilution. Membrane was washed twice with PBST for 5 and 10 min. Thereafter, GUS signals were detected using ECL Prime western blotting detection kit (GE Healthcare Biosciences, UK) as per manufacturer's protocol. Equal amount of protein loading was assured with Ponceau‐S staining.

CC patient and control tissues were collected from the Affiliated Kunshan Hospital of Jiangsu University. The patient tissues were preserved in liquid nitrogen and lysed using RIPA lysis buffer (Strong) (MedChemExpress, China). A BCA Protein Quantification Kit (Vazyme, China) was used to measure the protein concentration. Proteins were transferred to nitrocellulose membranes (Millipore, Billerica, MA, USA) after separation through 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis. Then, the membranes were incubated with primary antibodies against the following: NUP107/SEC13/ALDH7A1/ALG1/CHPF/FAM162A/FBP2/GALK1/IDH1/TGFA/VLDLR/XYLT2/OGDHL (Abcam, USA, 1:1000) and GAPDH (Abcam, USA, 1:3000). The membranes were washed and incubated with horseradish peroxidase-labeled goat anti-rabbit IgG (1:5000; Sigma). The signals were visualized using BIO-RADXR.

Cells were grouped and pretreated as previously described in the FCM. The total protein was extracted, separated using SDS-PAGE and transferred to a nitrocellulose membrane. The membrane was blocked with 5% skimmed milk at room temperature for 2 h, and then stained with rabbit polyclonal anti-B-cell lymphoma 2 (Bcl-2; 1:200; BA0412) or mouse monoclonal anti-β-actin antibodies (1:200; BM0627; Wuhan Boster Biotechnology Co., Ltd., Wuhan, China) for 2 h at room temperature. Following washing, the membrane was incubated with horseradish peroxidase-labeled goat-anti-rabbit IgG (Sigma-Aldrich, St. Louis, MO, USA) at 1:1,000 for 1 h at room temperature. The membrane was then washed and developed. All experiments were repeated three times.