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Fig. 2. Tbdn co-immunoprecipitation with cortactin. (A) The top panels show immunoprecipitation (IP) of IEM endothelial cell protein extracts using affinity purified anti-Tbdn rabbit antibody C10-20 (Tbdn), pre-immune rabbit serum (PreImm), monoclonal anti-cortactin antibody (4F11), negative-control IgG (Control) or affinity purified anti-cortactin antibody (SC11408) followed by western blotting with anti-Tbdn antibody C10-20. The blots shown in the bottom panels are the same blots as those shown in the top panels after the blots were stripped and re-probed using the anti-cortactin monoclonal antibody 4F11. Tbdn and cortactin are indicated by arrows at 100 kDa and 80 kDa, respectively. (B) Specificity of the presence of cortactin in Tbdn immunoprecipitates is demonstrated by immunoprecipitation of IEM endothelial cell protein extracts by using affinity purified anti-Tbdn antibody C10-20 (Ab: Tbdn) alone or in the presence of Tbdn C10-20-blocking peptide (Peptide: Tbdn) or control peptide (Peptide: Ctr) followed by western blotting with monoclonal anti-cortactin 4F11 antibody. PreImm, rabbit pre-immune serum. Whole-cell lysate (WCL) prepared from IEM cells was included as a positive control. An empty lane separates the IP and WCL lanes. (C) IP of protein extracts from IEM and RF/6A endothelial cells as indicated using: affinity purified anti-Tbdn antibody C10-20 (Tbdn), rabbit pre-immune serum (PreImm) or no-antibody control (Control) followed by western blotting with monoclonal anti-cortactin 4F11 antibody. Whole-cell lysates of IEM (IEM WCL), RF6A (RF/6A WCL), mouse 3T3 cells (3T3 WCL) are included as positive controls. Empty lanes are indicated (–). Representative experiments are shown.
