Fig. 4. PKC and Abl are both required for the response to ER stress. Neuro2a cells were transfected with control siRNA, Abl siRNA or PKC siRNA. After 48 hours, cells were pretreated with V1-1 (1 µM) for 15 minutes followed by Tm (5 µg/ml) treatment. (A) Total-cell lysates were analyzed by western blotting to confirm knockdown of Abl (upper panel) and PKC (lower panel). GAPDH in total lysates was used as internal loading control. (B) (Top two panels) Levels of PKC were analyzed by western blotting in the ER (upper panel) and mitochondrial (middle panel) fractions at the indicated time. (Lower panel) Histogram depicting the amount of PKC associated with the ER or mitochondria in Neuro2a cells. Data are expressed as the mean ± s.e. of three independent experiments. ^*P<0.05 vs Tm treatment, ^#P<0.05 vs Tm treatment in cells transfected with control siRNA. (C) Representative confocal images of PKC (red) and PDI (green) following 3 minutes of Tm treatment in the cells transfected with control or Abl siRNA. The data are from three independent experiments. Original magnification was x60. (D) Levels of Abl in mitochondrial fractions were analyzed by western blot after 3 hours of Tm treatment in cells transfected with control siRNA or PKC siRNA. Shown are representative data of three independent experiments. (E) Cell lysates from the ER fractions were subjected to immunoprecipitation (IP) with anti-PKC. In vitro kinase assays were carried out with or without V1-1 or knockdown of Abl using histone as a substrate. Data are representative of two independent experiments. (F) Cell lysates from the ER fractions were subjected to immunoprecipitation (IP) using anti-PKC antibody. The immunoprecipitates were analyzed by immunoblotting (IB) using anti-P-Tyr and anti-PKC antibodies. Data are representative for three independent experiments.