Supplementary Figure 7: Control experiments for in utero electroporation studies

a Western blot analysis of Nischarin in Neuro2A cells transfected with a shmiRNAs targeting Nischarin (shmiNISCH) or a scrambled negative control (shmiNEG). Cropped images are shown. b Quantification revealed a 60% reduction in Nischarin protein levels following transfection with a shmiNISCH (n = 3 flasks, one-tailed Mann Whitney test; P = 0.05). c-h Representative images for in utero electroporation experiments. Constructs were electroporated at E14.5 before analysis at E17.5. GFP positive cells were quantified in 3 blinded sections per animal in 9 areas across the developing cortex and expressed relative to the total number of cells. e, h Electroporation of a knock-down construct targeting Nischarin c-e or of a phosphomimetic mutant of Pak1(T422E) f-h in wild-type animals did not significantly influence neuronal migration (two-way repeated measures ANOVA with a Bonferroni multiple comparison test; shmiNISCH: n = 4 animals per condition, interaction P = 0.7074; Pak1(T422E): n = 5 animals per condition, interaction P = 0.3652). i-n Representative images of mouse brains electroporated with either Nischarin or pCAGEN control vector followed by immunostaining with sera targeting Nischarin (n = 3 animals per genotype). i-k Cells with highest levels of Nischarin congregated in the subventricular and intermediate zones. l-n No immunoreactivity for Nischarin was found in mouse brains electroporated with the control vector when employing those imaging conditions used in i-k. Note that these imaging conditions do not allow detection of the endogenous protein. Error bars show mean +/- standard error of the mean. The scale bar shows 100 μm in c, f, i and l.