Mouse anti human dek

The CRC cell line, SW-620, was grown on coverslips to 70% confluence, and then fixed in 4% paraformaldehyde for 10 min and permeabilized with 0.5% TritonX-100 for 10 min after 24 h. Blocking was performed with 3% bovine serum albumin fraction V (Solarbio, Beijing, China) for 1 h at room temperature. After washing with PBS, cells were incubated with mouse anti-human DEK (1∶50, BD Biosciences Pharmingen, San Diego, CA, USA) at 4°C overnight, followed by incubation with Alexa Fluor 568 goat anti-mouse IgG (H+L) (A11004, 1∶1000, Life Technologies, Carlsbad, CA, USA) for 1 h at room temperature. After washing with PBS, cells were counterstained with 49-6-diamidino-2-phenylindole (DAPI) (Beyotime, Shanghai, China) and the coverslips were mounted with Antifade Mounting Medium (Beyotime, Shanghai, China). Finally, immunofluorescence signals were visualized and recorded using Leica SP5II confocal microscope [21] (link).

Total cell lysates were prepared in RIPA buffer (150 mM NaCl, 1% NP40, 0.5% Sodium Deoxycholate, 0.1% SDS, 50 mM Tris-HCl, pH:8) containing protease and phosphatase inhibitors (ThermoFisher Scientific, Asheville, NC) followed by sonication (2 x 15 seconds). The lysates were separated on a 10% TRIS-HCL polyacrylamide gel (Biorad, Hercules, CA) and transferred to nitrocellulose membranes (Biorad). The membranes were incubated overnight at 4°C with mouse anti-human DEK (1:1000) (BD Biosciences) or with rabbit anti human GAPDH (1:5000) (CST) for 1 hour at room temperature, followed by incubation with horseradish peroxidase (HRP) conjugated secondary antibodies (CST). Protein bands were visualized with chemiluminescence (ThermoFisher Scientific,) using the ChemiDoc imaging system (Biorad).