Fig. 3. ER stress induces the interaction of PKC and Abl. Neuro2a cells were pretreated with V1-1 or TAT control (1 µM) for 15 minutes followed by Tm (5 µg/ml) treatment at the indicated time points. ER and mitochondrial (mito) fractions were then isolated. (A) The levels of Abl in the ER and mitochondria were detected by western blot. (Top) Graph depicting the levels of Abl 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 mitochondria fractions. (Bottom) Representative western blot of staining for Abl in ER and mitochondrial fractions of two independent experiments. Calnexin (ER marker) and TOM20 (mitochondrial marker) were used as loading controls. (B) (Top) ER and mitochondrial fractions were subjected to immunoprecipitation (IP) with anti-Abl antibody and the immunoprecipitates were analyzed by immunoblotting (IB) with anti-PKC and anti-Abl antibodies at the indicated time points. Shown are representative data of four independent experiments. (Bottom) Immunoprecipitates obtained using anti-PKC antibody were analyzed by immunoblotting (IB) using anti-Abl and anti-PKC antibodies at the indicated time points. Shown are representative data of two independent experiments. (C) ER fractions were isolated from the penumbra area of rats brains that had been subjected to 2 hours MCAO followed by 24 hours of reperfusion. Immunoprecipitates obtained using anti-Abl antibody were analyzed by immunoblotting with anti-PKC and anti-Abl antibodies. Data are representative of results obtained from five rats per group.