Fig. 3

Rescue of Munc18–1-knockdown-induced migration defects. a Characterization of an RNAi-resistant mMunc18–1, mMunc18–1R. pCAG-Myc-mMunc18–1R was cotransfected into COS7 cells with pSuper-H1.shLuc (Cont) or shMunc#1. Western blotting analyses were done as Fig. 2a. b pCAG-GFP was electroporated with pSuper-H1.shLuc + pCAG-Myc (i), with sh-Munc#1 + pCAG-Myc (ii) or sh-Munc#1 + pCAG-Myc-mMunc18–1R (iii) into cerebral cortices at E14.5, followed by fixation at P2. Coronal sections were stained for GFP (green) and nuclei (blue) (i-iii). Bar, 100 μm. Myc-tagged Munc18–1-R expression in (iii) was confirmed (lower panel). Bar, 5 μm. c Quantification of the distribution of neurons in distinct regions for each condition in (b). Error bars indicate SD (i, n = 5; ii, n = 4; iii, n = 7); ** p < 0.01; * p < 0.05, by Tukey-Kramer LSD. d Expression of Myc-Munc18–1 (WT) and Myc-tagged mutants (C180Y, R406H, M443R, G544 V) in COS7 cells. After 48 h of transfection, cells were harvested and subjected to western blotting (10% gel) with anti-Myc. Anti-Sept11 was used for a loading control