Fig. 4

Nicastrin is a membralin-dependent ERAD substrate. a Endogenous co-IP of nicastrin with membralin. Mouse brain organelle-enriched (nuclear and cytosol-free) lysates were precipitated with an anti-membralin antibody and immunoblotted with anti-nicastrin and membralin antibodies as indicated. A lower molecular weight band (~ 75 kDa) in the nicastrin blot was the brain organelle-enriched lysates treated with PNGase F to remove the glycosylation of nicastrin, serving as unglycosylated control. Untreated sample was used for endogenous co-IP, and shown in the input. b Western blot analysis and quantification of nicastrin protein levels detected in neuronal tissue from Mem +/+(n = 4 mice) and Mem−/− (n = 5 mice) animals. Data represent mean ± s.e.m. unpaired t-test, **P < 0.01. c Western blot analysis of nicastrin protein levels measured in Mem+/+or Mem−/− mouse NPCs treated with 10 µg/ml CHX for the indicated time. Data represent mean ± s.e.m. from three independent experiments, two-way ANOVA, **P < 0.01. d Native PAGE western blot analysis and quantification of γ-secretase subunits (nicastrin, PEN2 and PS1-NTF) in brains from Mem+/+(n = 4 mice) and Mem−/− (n = 5 mice) animals normalized to β-actin levels from an equivalent quantity of denatured lysate. Data represent mean ± s.e.m. unpaired t-test, *P < 0.05. e Western blot analysis and quantification of NICD protein levels in Mem+/+ and Mem−/− mouse NPCs. Data represent mean ± s.e.m. from 3 independent experiments, unpaired t-test, * P < 0.05. f γ-secretase activity was analyzed by measuring Aβ40 and Aβ42 generation in purified membrane fractions derived from Mem+/+ (n = 4 mice) and Mem−/− (n = 5 mice) mouse brains. Data represent mean ± s.e.m. unpaired t-test, * P < 0.05, ** P < 0.01. g Golgi staining of the hippocampus of Mem+/+ and Mem−/− mice (postnatal 3-day-old, n = 3 mice per genotype). Scale bars, 600 µm (left), and 60 µm in the magnified images (right). Number of Golgi-staining⁺ neurons per hippocampus (n = 9 sections per genotype) and number of primary dendrites per cell were quantified (n = 30-32 neurons per genotype). Data represent mean ± s.e.m. unpaired t-test, ***P < 0.001