Disruption of subcellular localization of MT-HNE proteins in U937T cells. (A) Glycosylation of WT-HNE or MT-HNE in U937T cells. Whole cell lysates of U937T-WT-HNE or -MT-HNE cells after induction of transgene expression were incubated with endoglycosidase H (H) or N-glycosidase F (F) or without enzyme (control; C) and were analyzed by immunoblot using anti-HNE (29-34 kDa). Resistance to digestion by endoglycosidase H indicates that HNE has reached the medial Golgi compartment. (B) Presence of HNE protein (29-34 kDa) in subcellular fractions of U937T cell lysates obtained by differential centrifugation: P1 (nuclear and nuclear-associated proteins), P2 (mitochondria, lysosomes, ER), P3 (plasma membrane, secretory and other vesicles, Golgi complex), and S (soluble cytosolic proteins). TL indicates total cell lysate. (C) Localization of cellular marker proteins in subcellular fractions obtained by differential centrifugation for WT-HNE and the mutants V72M and S97L. The nuclear pore complex protein nup 153 (190 kDa) defines P1. Lamp-2 (120 kDa) is a lysosomal protein specifying the lysosomal fraction (P2). To characterize fractions P3 and S, clathrin (180 kDa), which may be membrane associated or soluble, was used. Fractionation of the ER and the Golgi complex were defined with the use of calnexin (90 kDa) and GM 130 (130 kDa).