0 analysis software

Total protein extracts were obtained lysing cultured cells with ice-cold RIPA buffer (50 mM Tris-HCl pH 8.0, 150 mM NaCl, and 1% Nonidet P-40) supplemented with protease and phosphatase inhibitors (Sigma). Cellular extracts optimized to preserve DNMT enzymatic activity assay were obtained through the EpiQuik™ Nuclear Extraction Kit (EpiGentek Group Inc, NY, United States) following the manufacturer’s instructions. Purification of total histones was obtained according to the protocol provided by the Histone Extraction Kit (Abcam). Protein concentration was determined through the Bradford method, and samples were subjected to 10–12% SDS-PAGE. Gels were electroblotted into nitrocellulose membranes that were probed with the primary antibodies described above. Membranes were incubated with enhanced chemiluminescence (ECL) reagent solution (GE Healthcare, Hilden, Germany) and exposed to X-ray film (Santa Cruz). Immunoreactive band density was quantified using ImageLab v4.0 analysis software (Bio-Rad, Hercules, CA, United States).

Total protein extracts were obtained by lysing cultured cells with ice-cold RIPA buffer (50 mM Tris–HCl pH 8.0, 150 mM NaCl, 1% Nonidet P-40) supplemented with protease and phosphatase inhibitors (Sigma). Subcellular fractionation was performed using NE-PER^® Nuclear and Cytoplasmic Extraction Reagents (Thermo Scientific, Waltham, MA, USA, Pierce biotechnology), as previously described [12 (link)]. Protein concentration was determined using the Bradford method using bovine serum albumin as standard. In total, 10–30 μg of proteins was loaded and subjected to 8–12% SDS-PAGE, under reducing conditions. Gels were electroblotted into nitrocellulose membranes that were probed with the primary antibodies. Membranes were incubated with an enhanced chemiluminescence (ECL) reagent solution (GE Healthcare, Hilden, Germany) and exposed to X-ray film (Santa Cruz, Santa Cruz, CA, USA) or to Amersham Imager 600 (GE Healthcare). Immunoreactive band density was quantified with ImageLab v4.0 analysis software (Bio-Rad, Hercules, CA, USA) or with TotalLab Quant v2.2 software (TotalLab Ltd., Tyne, UK).

HIT WT cells and HIT-K185A cells were harvested in lysis buffer (Oetjen et al. 2007 (link)), and equal amounts of protein were subjected to SDS-PAGE and immunoblot analysis using antibodies against DLK (1:3000) (Oetjen et al. 2006 (link)) or (1:3000; GTX124127) (GeneTex, CA, USA), GAPDH (1:60,000; #sc-32233; 6C5) (Santa Cruz, Heidelberg, Germany) and α-Tubulin (1:1000; #2125) (Cell Signaling Technology, Beverly, MA, USA) were performed. The immunoreactive bands were visualized using ECL or ECLmax (for DLK detection in HIT-K185A cells) (BioRad Laboratories, München, Germany) and a chemiluminescence imaging system (ChemiDoc™ Touch Imaging System, BioRad Laboratories, München, Germany). Densitometric evaluation was performed using ImageLab 6.0 analysis software (BioRad Laboratories, München, Germany). To determine DLK half-time in the HIT WT and HIT-K185A cell line, cells were treated with cycloheximide (5 µg/ml, dissolved in DMSO) (Sigma-Aldrich, Steinheim, Germany) at the indicated time points before harvest. When indicated, cells were transiently transfected with expression vectors for DLK wild-type or its mutants.