Horseradish peroxidase conjugated mouse anti rabbit secondary antibody

Tumor samples were solubilized in radioimmunoprecipitation assay (RIPA) buffer for 30 minutes at 4°C with shaking for protein extraction and the protein was quantitated by the BCA protein assay (Invitrogen, CA, USA). Equal amounts of protein (40 μg) from the different groups were subjected to electrophoresis on a 10% sodium dodecyl sulphate (SDS) polyacrylamide gel, then transferred onto nitrocellulose membranes. The membranes were blocked with 5% BSA for 2 hours, then incubated at 4°C overnight with primary antibodies PNO1 (1: 800, catalog numbers: ab163419) (Abcam, Cambridge, United Kingdom) or AKT, mTOR, COX2 (1: 1000, Cell Signaling Technology, MA, USA) and GADPH (1: 2000, Cell Signaling Technology, MA, USA). GAPDH was used as the loading control. Membranes were washed 3 times with PBS (5 minutes each), then incubated with horseradish peroxidase-conjugated mouse anti-rabbit secondary antibody (1: 10000, catalog numbers: sc2357) (Santa Cruz Biotechnology, CA, USA). Membranes were visualized by chemiluminescence and using Image Lab software 4.1 (Bio-Rad, CA, USA) for densitometry analysis.

AOM (A5486), hemin (51280), EGCG (E4143), trigonelline hydrochloride (T5506), and MitoTempo (SML 0737) were obtained from Sigma-Aldrich, Inc. (St. Louis, MO, USA). DSS with a molecular weight of 36,000–50,000 was obtained from MP Biomedical Inc. (Santa Ana, CA, USA, MFCD00081551). The colorectal cancer cell line, Caco2, was purchased from the Korean Cell Line Bank (Seoul, Korea). Fetal bovine serum (FBS), phosphate-buffered solution (PBS), and high glucose Dulbecco’s Modified Eagle Medium (DMEM) were purchased from HyClone (Logan, UT, USA). Penicillin-streptomycin solution, cell culture dishes, and plates were purchased from Thermo Fisher Scientific Inc. (Waltham, MA, USA). CDK2 (sc-6248), CDK4 (sc-23896), cyclin D (sc-8396), cyclin E (sc-247), and β-actin (sc-47778) primary antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Nrf2 (NBP1-32822) and Keap1 (NBP2-03319) antibodies were purchased from Novus Biologicals (Centennial, CO, USA). Horseradish peroxidase-conjugated mouse anti-rabbit secondary antibody and -conjugated mouse IgG kappa binding protein were purchased from Santa Cruz Biotechnology. All mRNA primers were purchased from Cosmogenetech (Seoul, Korea).

Transfected A549 cells were washed twice in ice-cold PBS and lysed in RIPA lysis buffer with protease inhibitors (50 nmol/l HEPES, pH 7.5, 150 nmol/l NaCl, 1% glycerol, 1% Triton, protease inhibitor cocktail). The protein concentration was determined using a BCA Protein Assay kit (Beyotime Institute of Biotechnology). A total of 25 µg protein was loaded per sample for SDS-PAGE (12% acrylamide) and transferred to polyvinylidene difluoride membranes (EMD Millipore). The membranes were blocked using 5% skimmed milk in TBS-Tween (TBST; 0.05% Tween-20) at room temperature for 1 h, and then incubated with antibodies against VEGF (1:800; cat. no. 34-4300; Sigma-Aldrich; Merck KGaA), CCR-7 (1:2,000; cat. no. ab32527; Abcam) or tubulin (1:2,000; cat. no. SC-12462; Santa Cruz Biotechnology, Inc.) for 2 h at room temperature, the membrane was washed 3 times with TBST (10 min each), followed by incubation with horseradish peroxidase-conjugated mouse anti-rabbit secondary antibody for 1 h at room temperature (1:1,000; cat. no. sc-2357; Santa Cruz Biotechnology, Inc.). The signal was captured using a Super Signal West Pico chemiluminescent substrate (cat. no. 34080; Pierce; Thermo Fisher Scientific, Inc.) to visualize the bands, and Image-Pro Plus 6.0 software (Media Cybernetics, Inc.) was used for grayscale analysis.