Western Blot Analysis of Nuclear and Cytoplasmic Proteins

Nuclear or cytoplasmic extracts were prepared by the use of NE-PER Nuclear and Cytoplasmic Extraction Reagents (Pierce, Rockford, IL, USA) according to the manufacturer’s instructions. Protein concentration was determined by a DC protein assay kit (Bio-Rad, Richmond, CA, USA). Proteins were separated by SDS-PAGE and transferred to PVDF membranes (Immobilon-P, Millipore, Bedford, MA, USA). These membranes were probed with anti-WDR35 peptide antibody (amino acids 459–473, 1∶500), which was designed, produced, and purified by Medical & Biological Laboratories (Nagoya, Japan), or with antibodies against c-Jun, phospho-c-Jun (Ser73) (P-c-Jun), phospho-IκB-α (P-IκB-α), NF-κB (p65), α-tubulin, or nucleolin (Cell Signaling Technology, Danvers, MA, USA; 1∶1000). Detection was performed with the Western blotting reagent ECL Prime (GE Healthcare, Buckinghamshire, UK). Protein levels were quantified by densitometric scanning with the Gel-Pro Analyzer (Media Cybernetics, Inc., USA) and expressed as the ratio to α-tubulin or nucleolin levels as described previously [4] (link), [14] (link).