Horseradish peroxidase conjugated anti rabbit antibody

The protein lysate was used to extract lung tissue and mesenteric lymph node protein and determine the concentration. After sodium dodecyl sulphate-polyacrylamide gel electrophoresis, the protein was transferred to the polyvinylidene fluoride membrane by electrophoresis. The membranes were blocked with 5% skimmed milk and then incubated with primary antibodies (HMGB1 1:1000, RAGE 1:800, NF-kB p65 1:1000) (Cambridge, UK; Ab18256, Ab3611, Ab16502) and (GAPDH 1:1500, H3 1:1500) (Danvers, MA, USA, #5471, #8173) overnight at 4 °C. The membranes were incubated with a horseradish peroxidase-conjugated anti-rabbit antibody (1:1000, Beyotime, Shanghai, China, Ab18256) at 25 °C for 1 h. After the membrane is washed with eluent, the immunoreactive bands were visualized using ECL Plus (Life Technology Corporation, Gaithersburg, MD, USA) with a ChemiDoc XRS bioimaging system (Bio-Rad Laboratories, Inc., Hercules, CA, USA).

Cells were lysed using radioimmunoprecipitation buffer (Beyotime, Shanghai, China). Total protein (20 μg) was separated by 8% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred onto 0.45 μm polyvinylidene fluoride membranes (Millipore, Billerica, USA) using a Mini-Protean System (Bio-Rad, Hercules, USA). Membranes were incubated with primary antibody (1:500 dilution, catalog no. ABS1508, anti-SREBP1 antibody from Sigma-Aldrich; 1:500 dilution, catalog no. 3189S, anti-FASN antibody from Cell Signaling, Danvers, USA; 1:1 200 dilution, catalog no. AA128, anti-ACTB antibody from Beyotime) overnight at 4°C, then incubated with horseradish peroxidase-conjugated anti-rabbit antibody from Beyotime (1:8 000 dilution, catalog no. A0208) or anti-mouse antibody from Beyotime (1:12,000 dilution, catalog no. A0216) for 2 h at 37°C. Enhanced chemiluminescence (Beyotime) was added to the membranes, which were detected using an Odyssey Fc Imaging System (Li-Cor, Lincoln, USA) [22 (link)].

Western blot analysis procedure was described previously [55 (link)]. The primary antibodies were listed as follows: GAPDH (1:3000, Genesci, Shanghai, China); TET1 (1:500, GeneTex, California, USA); H3K9me2 (1:1000; Sino Biological Inc., Beijing, China), H3K27me3 (1:1000; Sino Biological Inc., Beijing, China), and OCT4 (1:500; Chemicon, Massachusetts, USA). Horse-radish peroxidase-conjugated anti-rabbit antibody (1:1000, Beyotime, Beijing, China) or anti-mouse antibody (1:2000, Beyotime, Beijing, China) was used as secondary antibodies.