FIGURE 3:

Cdo and Stim1 form complexes in 293T cells and differentiating C2C12 cells, and netrin-2 enhances this interaction. (A) 293T cells were transfected with SRT-tagged Stim1, Cdo, or control (−) expression vectors, and 48 h later, lysates were immunoprecipitated with Cdo antibodies and immunoblotted with antibodies for SRT or Cdo. (B) Lysates of 293T cells transfected with indicated vectors were subjected to immunoprecipitation with an antibody to a SRT tag and immunoblotting with antibodies for SRT or Cdo. (C) Lysates of C2C12 cell cultures from G, DM1, or DM2 were immunoprecipitated with Stim1 antibodies and immunoblotted with antibodies for Cdo or Stim1 (left). Lysates of C2C12 cells at DM1 were immunoprecipitated with control rabbit IgG or Stim1 antibodies and immunoblotted with Cdo and Stim1 (right). (D) Lysates of C2C12 cell cultures from G, DM0, or DM1 were immunoprecipitated with Cdo antibodies and immunoblotted with antibodies for Cdo or Stim1 (left). As control, lysates of C2C12 cells at DM1 were immunoprecipitated with control rabbit IgG or Cdo antibodies and immunoblotted with Cdo and Stim1 (right). (E) Confluent C2C12 myoblasts were incubated for 8 h in DM and treated with netrin-2 for indicated times. Lysates were subjected to immunoprecipitation with Cdo antibodies and immunoblotting with indicated antibodies. p-NFATc3 and NFATc3 signals were quantified by densitometry. The signal ratio of C2C12 cells without netrin-2 was set to 1 (left). As control, lysates of C2C12 cells treated with netrin-2 for 60 min were immunoprecipitated with control rabbit IgG or Cdo antibodies and immunoblotted with Cdo and Stim1 (right). (F) Control (top; the red boxed areas in a–c are shown in a′–c′) and Cdo-depleted (bottom; the red boxed areas in d–e are shown in d′–e′) C2C12 cells transfected with Stim1-RFP were treated with netrin-2 for the indicated times, and live confocal images were taken. The TG (1 μM) treatment of the Cdo-depleted cell serves as the positive control.