Ecl detection system

Cells were fixed in 4% paraformaldehyde, post‐fixed using absolute methanol for 5 min, and stained according to previously published protocols (Mattioli et al., 2011). Primary antibodies were applied overnight at 4°C, and secondary antibodies were used for 1 hour at room temperature. Nuclei were counterstained with 4,6‐diamidino‐2‐phenylindole (DAPI). Sample observation and image acquisition were performed using a Nikon Eclipse Ni epifluorescence microscope equipped with a digital CCD camera and NIS‐ Elements 4.3 AR software. Photoshop CS and Photoshop 7 were used for image processing. Mean fluorescence intensity (MFI) was measured using NIS‐ Elements 4.3 AR.
For Western blot analysis, tissues and cells were lysed in buffer containing: 20 mM Tris‐HCl (pH = 7.5), 1% SDS, 1 mM Na3VO4, 1 mM PMSF, 5% beta‐mercaptoethanol, and protease inhibitors. Proteins were subjected to SDS gradient gel (5%–20%) electrophoresis and transferred to nitrocellulose membrane overnight at 4°C. After incubation with primary and secondary antibodies, immunoblotted bands were revealed by Invitrogen ECL detection system. Densitometry was performed by a Bio‐Rad GS800 Densitometer equipped with Quantity One Software. Densitometric values were normalized to corresponding GAPDH bands if not differently stated.

To measure phosphorylated proteins, S3-GFP and R4-GFP cells were treated with IFN-α or IFN-λ for 30 minutes before harvesting. Cells were harvested by the treatment of trypsin-EDTA. Cells were lysed in ice-cold lysis buffer (50mM Tris HCl pH 8.0, 140 mM NaCl, 1.5 mM MgCl2, 0.5% NP-40 with complete protease inhibitor from Invitrogen) for 10 minutes in ice (about 1X10⁶ cells/200 μL). Whole cells and cell debris were pelleted by low speed centrifugation and cleared supernatants were transferred to a new tube. Protein concentration was determined by Nanodrop (Thermo Scientific). Samples were boiled for 10 minutes at 80°C in the presence of 1X SDS-PAGE-loading buffer (250mM Tris-HCL pH 6.8, 40% glycerol, 8% SDS, 0.57M β-mercaptoethanol, 0.12% bromophenol blue). 20μg of protein was loaded onto 12% SDS-PAGE and transferred into a nitrocellulose membrane (Hybond, Amersham Biosciences). The membrane was blocked using 5% blotting-grade milk powder (Biorad, Hercules, CA), for one hour then incubated with primary antibody. After overnight incubation, the antigen-antibody complex was visualized with HRP-conjugated goat anti-rabbit or anti-mouse IgG and the ECL detection system (Invitrogen, Pierce, Amersham).

6mA in genomic DNA was measured by ELISA and dot-blot assay. ELISA was performed as in our previous report [17 (link), 18 (link)]. The MethylFlash 6mA DNA Methylation ELISA Kit (Colorimetric) (EpiGentek, NY) was used according to the manufacturer’s instructions. Each sample was run in triplicate. For dot-blot assays, denatured genomic DNA (2 µl, 100 ng) was spotted onto a nylon membrane, UV-crosslinked (20 s, 1200 J/cm²), and blocked in 5% milk for 1 h at room temperature (RT). Then, the membranes were incubated with the 6mA antibody for 4 h, and the secondary antibody for another 1 h at RT. After washing, and the dots was developed and visualized using an ECL detection system (Invitrogen) according to the manufacturer’s protocol. The staining of membranes with methylene blue confirmed the equal DNA loading.

Whole cell lysates were prepared from cultured cells and melanoma tissues. Standard western blotting assay was performed as previously described^{41 (link)}. Immunoreactive bands were visualized using the Enhanced chemiluminescence (ECL) detection system (Invitrogen, Carlsbad, CA, USA).

Transfected HEK293 cells were subjected to IP lysis buffer containing: 50mM Tris-HCl (ph = 8), 150 mM NaCl; 1% NP-40; 1.5 mM MgCl; 1 mM DTT; 1 mM PMSF; 20 mM NaF; 0.05% SDS; phosphatases and proteases inhibitors. After sonication, clarification and protein quantification, 700 μg of lysate for samples were immunoprecipitated with 1 μL/mL of anti-FLAG antibody (Sigma, St.Luis, MO, USA) over-night at 4 °C. After the addition of 30 µL/mL of protein A/G (Santa Cruz, Dallas, TX, USA) for 60 min at 4 °C, the immunoprecipitated proteins were washed with lysis buffer 3 times. Subsequently, immunoprecipitated samples were added to Laemmli’s buffer and subjected to immunoblotting analysis. Incubation with primary and secondary antibodies was performed and immunoblotted bands were revealed by Invitrogen ECL detection system. Densitometric analysis was performed with ImageJ2 program.