Detecting Post-Translational Modifications in YB-1 Protein

To detect the post-translational modification of YB-1 protein, two-dimensional (2D) WB was conducted as previously described [45 (link)]. Whole cell lysate of iDC and 27DC were prepared using RIPA (Boston BioProducts, Milford, MA, USA) supplemented with 5 mM EDTA (Quality Biological), 1X protease inhibitors (MilliporeSigma), and 1X phosphatase inhibitors cocktail (Thermo Fisher Scientific) at 10x10⁶ cells/mL for 15 minutes on ice. The cell lysate was centrifuged at 15,000 x g for 10 minutes at 4°C to remove cell debris. Protein concentration was determined using a BCA Assay. The total cellular protein (100 μg) was subjected for 2D-WB analysis [45 (link)].