Increased expression of Dkk1 by HBZ. (A) Expression of HBZ wt and HBZ-MutAD in the stable cell lines. Total cellular lysates (50 μg) were subjected to Western blot analysis using antibodies directed against Myc (C-terminal epitope tag on HBZ) and β actin, as indicated. (B) Levels of Dkk1 mRNA in cells expressing HBZ wt, HBZ-MutAD, or carrying the empty vector. Levels of Dkk1 mRNA were normalized to UBE2D2 mRNA following quantitative real-time PCR of reverse transcribed total cellular RNA. The graph shows data from three independent RNA extractions, with Dkk1 mRNA levels shown relative to values obtained from cells containing the pcDNA3.1 empty vector (set to 1). (C) Levels of Dkk1 protein in the culture medium from cells expressing HBZ wt, HBZ-MutAD, or carrying the empty vector. Acid-precipitated proteins from culture media of indicated cell lines were resolved by SDS-PAGE and subjected to Western blot analysis using an antibody directed against Dkk1 (upper panel) or stained with Coomassie blue (lower panel). (D) Inhibition of Dkk1 glycosylation in cells expressing HBZ wt, HBZ-MutAD, or carrying the empty vector. The indicated cell lines were treated with DMSO (vehicle) or tunicamycin, as denoted, and acid-precipitated proteins from the culture media were analyzed by Western blot using an antibody directed against Dkk1.