Figure 4

RTN1-C induces mitochondrial-associated apoptosis during OGD/R. (a) Representative western blot analysis of cytosolic cytochrome C (cyt-Cyto C), mitochondrial cytochrome C (mito-Cyto C) and cleaved-caspase-3 in N2a cells exposed to OGD/R treatment or normal condition, after transfection with RTN1-C-FLAG or shRTN1-C. GAPDH was used as a loading control. Fractionation experiment was performed before western blot. (b) Histograms showing quantification of images in a. The results were normalized to COX IV or GAPDH expression, and the quantitative data were expressed as the mean±S.D. on different assays (n=3). *P<0.05; **P<0.01; ***P<0.001. (c) Representative western blot analysis of mitochondrial and cytosolic Bcl-xL in N2a cells transfected with RTN1-C-FLAG or empty vector for 24 h, then the cells exposed to OGD/R treatment or normal condition. COX IV and GAPDH were used as the markers of mitochondria and cytosol, respectively. (d) Histogram showing quantification of images in c. The results were normalized to COX IV or GAPDH expression, and the quantitative data were expressed as the mean±S.D. on different assays (n=3). *P<0.05; **P<0.01; ***P<0.001. (e) Representative western blot analysis of mitochondrial and cytosolic Bcl-xL in N2a cells transfected with shRTN1-C or shNC for 24 h, then the cells exposed to OGD/R treatment or normal condition. COX IV and GAPDH were used as the markers of mitochondria and cytosol, respectively. (f) Histogram showing quantification of images in e. The results were normalized to COX IV or GAPDH expression, and the quantitative data were expressed as the mean±S.D. on different assays (n=3). *P<0.05; **P<0.01. (g) Immunoprecipitation of endogenous Bcl-xL and RTN1-C. N2a cells were treated with OGD/R and the lysates were incubated with anti-RTN1-C antibody. Bcl-xL and RTN1-C were detected by immunoblotting with anti-Bcl-xL and anti-RTN1-C antibody, respectively. (h) Representative western blot analysis of mitochondrial and cytosolic Bcl-xL in N2a cells exposed to OGD/R treatment after transfection with RTN1-C-FLAG or shRTN1-C. COX IV and GAPDH were used as the markers of mitochondria and cytosol, respectively. (i) Histogram showing quantification of images in h. The results were normalized to COX IV or GAPDH expression, and the quantitative data were expressed as the mean±S.D. on different assays (n=3). *P<0.05