Fig. 1

Role of mRbfox1-iso2 in neuronal migration during mouse brain development. a Characterization of RNAi vectors. pCS-MT-mRbfox1-iso1 or pCS-MT-mRbfox1-iso2 (Myc-iso1 or Myc-iso2) was transfected into COS7 cells with pSuper control vector (vector), pSuper-mRbfox1-iso1, pSuper-mRbfox1-iso2 or pSuper-mRbfox1-iso1/2. After 48 h, cells were harvested and subjected to western blotting (20 μg protein per lane) with anti-A2BP1(Rbfox1). Anti-Sept11 was used for loading control. The values represent relative density of the bands normalized to Sept11 with ImageJ software. The band intensity of the control experiments was defined as 1. b Migration defects of mRbfox1-iso2-deficient cortical neurons. pCAG-EGFP was electroporated in utero with pSuper control vector, pSuper-mRbfox1-iso2 (iso2), or pSuper-mRbfox1-iso1/2 (iso1/2) into cerebral cortices at E14.5. Coronal sections were prepared at P3 and immunostained for GFP (white) and DAPI (blue). Bar, 100 μm. c Quantification of the distribution of transfected neurons in distinct parts of the cerebral cortex (bin 1–5 and IZ) for each condition shown in b. Error bars indicate SD; control (n = 5), iso2 (n = 5), iso1/2 (n = 3). **p < 0.01 *p < 0.05 by Tukey-Kramer LSD. d Characterization of an RNAi-resistant version, mRbfox1-iso2R. pCAG-Myc-mRbfox1-iso2 (Myc-iso2) or pCAG-Myc-mRbfox1-iso2R (Myc-iso2R) was transfected into COS7 cells with pSuper control vector or pSuper-mRbfox1-iso2. After 48 h, cells were harvested and subjected to western blotting with anti-Myc. The blot was reprobed with anti-Sept11. e Rescue experiments of mRbfox1-iso2 knockdown. pCAG-EGFP was coelectroporated in utero with pSuper control vector plus pCAG vector (control) or pSuper-mRbfox1-iso2 together with pCAG vector (empty) or pCAG-Myc-mRbfox1-iso2R (iso2R) into cerebral cortices at E14.5, followed by fixation at P3. Coronal sections were stained as in b. Bar, 100 μm. f Quantification of each condition shown in e. Analyses were done as in c. Error bars indicate SD; control (n = 5), iso2 (empty, n = 5), iso2 + iso2R (n = 5). **p < 0.01 by Tukey-Kramer LSD. g Positioning of mRbfox1-iso2-deficient neurons at E17.5, P7, and P16. In utero transfection was done as in b at E14.5, followed by fixation at the indicated time points. Coronal sections of control and knockdown samples were immunostained for GFP. Bars, 100 μm