Electroporated ADAM10 morpholinos induce donwregulation of endogenous ADAM10 protein during development of the spinal cord.

After electroporation at E4, the embryos were collected and transverse sections were cut at E6. The transfected cells are marked by green fluorescence. The control morpholinos and the untransfected side of the spinal cord serve as controls. The immune reactive cells are stained red. Cell nuclei are stained with 4′,6-diamidino-2-phenylindole (DAPI) as blue. (A–H) The expression of ADAM10 protein in the spinal cord transfected with control morpholinos (Fluo-m; A–D) or ADAM10 morpholinos (AM10-m; E–H). Arrows indicate transfected regions in the ventricular zone, and arrowheads in the mental layer. (I–L) Apoptotic cells (red, arrowheads) measured by TUNEL assay. (M, N) Representative Western blots (M) and semi-quantitative Western blot analysis (N) of ADAM10 protein, including a pre-mature (pADAM10) and a mature (mADAM10) form, in the transfected (tran) and untransfected side (untran) of ADAM10-morpholinos (AM10-m) or control morpholinos (Fluo-m) transfected embryos. GAPDH is used as a loading control. The amount of ADAM10 protein is normalized by the number of the control side, which is set to be 1. All data are presented as mean ± SEM from at least 3 independent samples (**p