Journal: Acta Neuropathologica Communications
Article Title: MUNC18–1 gene abnormalities are involved in neurodevelopmental disorders through defective cortical architecture during brain development
doi: 10.1186/s40478-017-0498-5
Figure Lengend Snippet: Effects of Munc18–1 knockdown on subcellular distribution of N-Cadherin. a Localization of exogenous N-Cadherin in Munc18–1-deficient migrating neurons. E14.5 cerebral cortices were electroporated with pCAG-RFP plus pCAG-HA-N-Cadherin together with pSuper-H1.shLuc (i) or sh-Munc#1 (ii, iii) . Coronal sections were prepared at E18.0 and immunostained for HA-tag (i, ii) or HA-tag plus GM130 (iii) . (c) . Bars in ( i - ii ), 10 μm and ( iii ), 5 μm. b Quantification of N-Cadherin accumulation at Golgi. The ratio of RFP-positive cells with the accumulation was calculated for migrating neurons in the lower CP in ( a ). Error bars indicate SD; Control (n = 5), Munc18–1-knockdown (n = 5); ** p < 0.01 by Tukey-Kramer LSD. c Quantification of subcellular localization of N-Cadherin. The ratio of N-Cadherin in perinuclear to other cytoplasmic regions was analyzed. Error bars indicate SD of 5 brains containing more than 200 cells. ** p < 0.01 by Student’s t -test. d Localization of endogenous N-Cadherin in Munc18–1-deficient cortical neurons. pCAG-GFP was coelectroporated with pSuper-H1.shLuc (Control) or sh-Munc#1 into the E14.5 cerebral cortices. Neurons were isolated at E16.5, cultured for 48 h, fixed and stained with polyclonal anti-N-Cadherin without permeabilization. Bar, 10 μm. e Quantification of fluorescence intensity profiles of cell surface N-Cadherin across the cell bodies of control (blue) and Munc18–1-deficient neurons (red). Means +/− SEM (Control, n = 6; sh-Munc#1, n = 7). f Quantification of fluorescence intensity profiles of cell surface N-Cadherin in neurites. After the staining as in ( d ), neurons were permeabilized and double-stained with monoclonal anti-N-Cadherin and anti-GFP. Then, the ratio of the fluorescent intensity of surface N-Cadherin to total N-Cadherin was analyzed. Error bars indicate SD in each condition (n = 4). More than 300 neurites were analyzed in each experiment. ** p < 0.01 by Student’s t -test
Article Snippet: Mouse monoclonal anti-Munc18–1, anti-Syntaxin1A, anti-nestin and anti-N-Cadherin antibodies were purchased from Abnova Inc. (H00006812-M01; Taipei, Taiwan), Synaptic Systems (110,111; Gottingen, Germany), R&D Systems (MAB2736; Minneapolis, MN) and BD Biosciences (610,920; San Jose, CA), respectively.
Techniques: Knockdown, Control, Isolation, Cell Culture, Staining, Fluorescence
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