Fig. 8

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