Proteasome inhibition leads to accumulation of N4BP1 at PML NBs. (A-F) MEFs treated with MG132 were examined by indirect immunofluorescence using anti-N4BP1 (A,C,D,F), anti-fibrillarin (B,C) and anti-PML (E,F), as labeled in each panel. (A-C) A significant increase in N4BP1 outside the nucleolus was found (arrows point to non-nucleolar N4BP1). (D-F) There is a comparable increase in the colocalization of N4BP1 with PML (arrows point to colocalized N4BP1 and PML). (G) Total protein extracts and detergent-soluble and -insoluble fractions were prepared from either untreated or MG132-treated MEFs, followed by immunoblotting (IB) with anti-N4BP1 to detect N4BP1 levels. MG132 treatment leads to accumulation in the detergent-insoluble fraction. Lower panels show reblotting with anti-β-actin to confirm equivalent sample loading.