Mutant H338R and G392E neuroserpin accumulate as polymers within the ER

() Confocal microscopy analysis of COS-7 cells cultured for 48 h after transfection with wild-type, H338R or G392E neuroserpin and stained for neuroserpin (red) and the ER-resident protein calreticulin or the Golgi-resident protein GM130 (green). Only the merged images are shown in which yellow colour corresponds to areas with overlapping red and green staining. The nucleus appears blue due to DNA staining with DAPI. Scale bar: 10 µm. () Endoglycosidase-H (eH) digestion of samples from cells transfected with wild-type or each mutant neuroserpin that were pulsed-labelled and chased for 6 h. Cells lysates (C) and culture media (M) were analysed by immunoprecipitation and 8% w/v SDS–PAGE. Arrow: fully glycosylated and secreted neuroserpin, 55 kDa; arrowhead: intracellular neuroserpin intermediate, 50 kDa; black and white arrow: deglycosylated intracellular neuroserpin, 45 kDa; the asterisk indicates a second band of extracellular wild-type neuroserpin due to extracellular proteolysis; the dash indicates a slower migrating band of extracellular G392E neuroserpin. () 7.5% w/v acrylamide non-denaturing PAGE and western-blot analysis for neuroserpin in cell lysates (C) and culture media (M) from COS-7 cells transfected with wild-type or mutant neuroserpin or with a control plasmid expressing luciferase (Lucif.). Cells were cultured for 72 h after transfection. Arrow: wild-type and S52R neuroserpin monomers; arrowhead: S49P neuroserpin monomer; curly bracket: neuroserpin polymers.<p><b>Copyright information:</b></p><p>Taken from "The intracellular accumulation of polymeric neuroserpin explains the severity of the dementia FENIB"</p><p></p><p>Human Molecular Genetics 2008;17(11):1527-1539.</p><p>Published online 11 Feb 2008</p><p>PMCID:PMC2387220.</p><p>© 2008 The Author(s)