Lumilight plus ecl substrate

The biotinylated samples were analysed by SDS-PAGE; the proteins were transferred onto a PVDF membrane and detected with streptavidin-horseradish peroxidase (SA-HRP; Pierce, USA) according to the manufacturer’s instructions. After incubation with SA-HRP, the membranes were washed 4 times for 15 min with TBST (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.1% (m/v) Tween-20) and the blots then developed using the Lumi-Light Plus ECL substrate (Roche, Switzerland), Kodak Biomax ECL films and Kodak developing solutions.

Western blots were performed essentially as described before 19 (link). In brief, cells were lysed in Laemmli lysis buffer and the lysates were incubated for 5 min. at 95°C. Afterwards, protein samples were separated by 10% SDS gel electrophoresis and transferred to a PVDF membrane (Millipore, Billerica, MA, USA). Membranes were blocked for 1 hr in 4% milk in TBST and incubated overnight with monoclonal antibodies against α-smooth muscle actin (a-SMA), tubulin, collagen (all Santa Cruz Biotechnology, Santa Cruz, CA, USA), phospho-ERK1/2 or total ERK1/2 (both Cell Signalling, Leiden, The Netherlands) at 4°C. All secondary antibodies were horseradish peroxidase (HRP)-conjugated from DakoCytomation (Glostrup, Denmark) and diluted according to the manufacturer's instructions. Blots were imaged using Lumilight plus ECL substrate from Roche (Almere, The Netherlands) on an ImageQuant LAS 4000 biomolecular imager from GE Healthcare (Buckinghamshire, UK).

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.

Fibroblasts were seeded in 12-well plates in DMEM supplemented with 10% FCS. Next, medium was removed and cells were washed with serum-free medium. After serum starvation for 4 hours, the cells were incubated with serum-free medium (negative control) or RAW264.7 conditioned medium with or without 10 μM of each TGF-β receptor inhibitor or 10 μM P1pal-12. When indicated, cells were pre-incubated with 10 μM P1pal-12 for 30 minutes. Twenty four hours later, cells were lysed in Laemmli lysis buffer and Western blots were performed as described before [14 (link)]. In brief, protein samples were separated by 10% SDS gel electrophoresis and transferred to a PVDF membrane (Millipore, Billerica, MA). Membranes were blocked for 1 hour in 4% milk in TBST and incubated overnight with monoclonal antibodies against a-SMA (1:1000, Santa Cruz, CA), GAPDH (1:1000, Santa Cruz, CA), collagen (1:800, SouthernBiotech, AL) or p-SMAD2 (1:1000, Cell Signaling Technology, Boston, MA) at 4°C. All secondary antibodies were horseradish peroxidase (HRP)-conjugated from DakoCytomation (Glostrup, Denmark) and diluted according to the manufacturer's instructions. Blots were imaged using Lumilight plus ECL substrate from Roche (Almere, The Netherlands) on an ImageQuant LAS 4000 biomolecular imager from GE Healthcare (Buckinghamshire, U.K).

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).