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Fig. 7 Amotl2 associates with and promotes peripheral translocation of c-Src. (A) HA-Amotl2 was coexpressed with Src-WT-GFP or Src-Y527F-GFP in HEK293T cells and their interaction was examined by reciprocal immunoprecipitation. (B) Overexpressed HA-Amotl2 associated with endogenous c-SRC in human HEK293T cells. HA-Amotl2 was immunoprecipitated with anti-HA antibody and the precipitates were examined by western blotting for the presence of c-Src (indicated by an arrow) with anti-c-Src antibody. TCL, total cell lysate. (C) Overexpressed HA-Amotl2 or Myc-Amotl2 was co-localized with overexpressed Src-WT-GFP (upper panel) or endogenous c-Src (lower panel) in HeLa cells. (D) Examination of Amotl2 association with related tyrosine kinases Fyn and Yes of zebrafish. Myc-Amotl2 was coexpressed with HA-Src, HA-Fyn or HA-Yes in human HEK293T cells. Reciprocal co-immunoprecipitation was performed. Note that Myc-Amotl2 was co-precipitated with HA-Src (indicated by arrows), but not with HA-Fyn or HA-Yes. (E) Effect of Amotl2 on translocation of exogenous active c-Src. HeLa cells were transfected with Src-Y527F-GFP alone, or in combination with Myc-Amotl2 or Myc-Amotl2ΔPDZ, and treated with bradykinin. Lower panel: high magnification of an area boxed in the corresponding cell in the upper panel. (F) Effect of Amotl2 on translocation of endogenous phosphorylated c-Src in HeLa cells. Following transfection with Myc-Amotl2 (upper panel) or Myc-Amotl2ΔPDZ (lower panel), cells were treated with bradykinin and stained with p-c-Src (Thr420) antibody. (G) Co-localization of Myc-Amotl2 with endogenous endosomal proteins RhoB (upper panel) and EEA1 (lower panel) in HeLa cells. Scale bars: 10 μm.