Horseradish peroxidase conjugated donkey anti goat igg

Briefly, total proteins were extracted in RIPA (radio immunoprecipitation assay) buffer (9806; Cell Signaling, Danvers, MA, USA) containing 1 mM phenylmethylsulfonyl fluoride (8553S; Cell Signaling, Danvers, MA, USA) according to the manufacturer’s protocol. The BCA Protein Assay Kit (HX18651; Hoaxing, China) was used to measure protein concentration. Electrophoresis was performed with 30 μg total proteins separated by 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membranes (IPVH00010, Millipore, USA). The membrane was blocked with 5% (w/v) nonfat dry milk in 0.05 M Tris-buffered saline and 0.1% Tween-20 (TBST, pH 7.4) for 1 h and incubated with anti-ISL1(1:300; AF1837; R&D, Minneapolis, MN, USA) antibody and internal control GAPDH antibody (1:10,000; Ambion, USA) overnight at 4 °C. The secondary antibody, horseradish peroxidase-conjugated donkey anti-goat IgG (1:10,000; ab205723; Abcam, Cambridge, MA, USA), was diluted 1:5000 in TBST. The membranes were visualized using the SuperSignal West Pico Kit (Thermo Scientific, Waltham, MA, USA) substrate at room temperature. We used the ImageJ Software to assay the relative intensity of each blot. The intensity values of each group were normalized to GAPDH (internal control) in the same group.

Lungs were obtained from rats at the end of experiment. Nuclear fraction was collected by using the CNM compartmental protein extraction kit (BioChain Inc., San Francisco, CA, USA) according to the manufacturer’s instruction. Twenty μg of protein was separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to nitrocellulose membrane. The membrane was incubated with primary antibody (DNMT1, 1:1000, purchased from Abcam, Cambridge, UK; DNMT3A, 1:300, purchased from Abcam, Cambridge, UK; DNMT3B, 1:1000, purchased from GeneTex, Irvine, CA, USA; Histone H3, 1:5000, purchased from GeneTex, Irvine, CA, USA) at 4°C for 24 h, and then incubated with horseradish peroxidase-conjugated donkey anti-goat IgG (Abcam, Cambridge, UK) or horseradish peroxidase-conjugated goat anti-rabbit IgG (Cell signaling Technology Inc, Danvers, MA, USA) at room temperature for 2 h. The protein levels were detected by the enhanced chemiluminescence Western blotting detection reagent (Thermo scientific, Rockford, IL, USA), and quantified by the ImageJ software version 1.46r (National Institutes of Health, Bethesda, MD, USA).