Horseradish peroxidase hrp conjugated secondary antibody against rabbit igg

Total protein was extracted from BEAS-2B cells, separated by 10% SDS gel electrophoresis and transferred to a PVDF membrane (Millipore, Billerica, MA). Membranes were blocked for 1 hour in 5% milk in tris-buffered saline with 0.1% Tween 20 (TBST) buffer (TBST) and incubated overnight with (primary) antibodies against phospho-NF-κB p65 (Ser536) (1: 1000, #3033; Cell Signaling Technology, Leiden, The Netherlands), NF-κB p65 (1: 1000, #3034N; Cell Signaling Technology, Leiden, The Netherlands), Dnmt3b (1:250, ab2851; Abcam, Cambridge, UK) or beta-actin (1: 1000, 4967L; Cell Signaling Technology) at 4°C. After incubation with horseradish peroxidase (HRP)-conjugated secondary antibody against rabbit IgG (1: 2000, #7074; Cell Signaling Technology) for 1 hour at room temperature, blots were imaged using Lumilight plus ECL substrate (Roche, Almere, The Netherlands) on an ImageQuant LAS 4000 biomolecular imager (GE Healthcare, Buckinghamshire, UK). For quantification, densitometry was performed with ImageJ (National Institutes of Health, Bethesda, MD; https://imagej.nih.gov/ij/) using the histogram function in a selected area of mean gray value for each band.

Western blot was performed as reported [30 (link)]. Briefly, total protein was extracted and separated by 10% SDS gel electrophoresis and transferred to a PVDF membrane (Millipore, Billerica, MA, USA). Membranes were blocked for 1 h in 5% milk in tris-buffered saline (TBS) supplemented with 0.1% Tween 20 (TBST buffer) and incubated overnight with (primary) antibodies against TET2 (ab94580; Abcam, Cambridge, MA, USA), or Beta-Actin (4967L, Cell Signaling Technology, Leiden, The Netherlands) at 4 °C. After incubation with horseradish peroxidase (HRP)-conjugated secondary antibody against rabbit IgG (#7074, Cell Signaling Technology), blots were imaged using Lumilight plus ECL substrate (Roche, Almere, The Netherlands) on an ImageQuant LAS 4000 biomolecular imager (GE Healthcare, Buckinghamshire, UK).