Fig. 1. PKC translocates to the ER following ER stress. (A) Neuro2a cells were treated with Tm (5 µg/ml). Cell lysates from the ER and mitochondria-enriched fractions were subjected to western blot analysis at the indicated times. (Top panel) Changes in the level of PKC in the ER and the mitochondrial fractions at the indicated time points. ^*P<0.05 vs control in ER fractions, ^#P<0.05 vs control in mitochondrial fractions (n=3). (Centre panel) Representative western blot of PKC in the two fractions. Calnexin and TOM20 (markers of ER and mitochondria, respectively) were used as internal loading controls for quantification. (Bottom panel) The relative purity of the cellular fractionations was determined by the presence of calnexin, TOM20 and enolase (cytosolic marker). (B) Neuro2a cells were treated with Tm (5 µg/ml) or Tg (3 µM). (Top panel) Representative confocal microscopy image of PKC (red) and PDI (green), demonstrating increased colocalization (yellow) of PKC in the ER following 3 minutes of Tm or Tg treatment. Original magnification was x60. The data are from three independent experiments. (Bottom panel) Representative confocal microscopy image of PKC (red) and mitotracker (green) after 3 minutes or 3 hours of Tm treatment. The data are from two independent experiments. (C) Rats were subjected to 2 hours MCAO followed by 10-240 minutes of reperfusion (I/R). Lysates from the penumbra area of the ipsilateral hemisphere of the rats brains were fractionated. (Top panel) ER fractions were subjected to western blotting using PKC antibody. Calnexin was used as an internal loading control for quantification. (Centre panel) Histogram depicting the amount of PKC associated with the ER in brain samples (PKC/calnexin). Data are expressed as the mean ± s.e. from results of four rats, ^*P<0.05; ^**P<0.01, vs sham-operated rats. (Bottom panel) Confirmation of purity of fractionations by using the specific protein markers as described in Fig. 1A. (D) ER localization of PKC determined by immunoelectron microscopy. Representative electron microscopy image of PKC staining in the ER fractions from brains subjected to 2 hours MCAO followed by 4 hours of reperfusion (I/R). (magnification x35,000). Samples were probed in the presence (+) or absence (–) of PKC antibody. Arrows indicate PKC-positive staining with gold particles. Quantification of gold particles associated with ER lumen are provided in the lower histogram. Five random fields of each section from three animals were counted. Data represent the mean ± s.e. from results of three animals per group, ^*P<0.05 vs sham-operated rats.