FIG. 2.

Interaction of DCC and Trio. (A) Lysates of HEK-293 cells transfected with pEGFP-Trio and pRK5-DCC were submitted to immunoprecipitation using anti-DCC antibodies (DCC) or mouse IgG coupled to protein G-Sepharose beads. Immunoprecipitated proteins and 10% of the total cell lysates were submitted to SDS-PAGE, and GFP-Trio and DCC were detected by Western blotting using anti-DCC and anti-GFP antibodies. (B) Lysates of E18.5 mouse brains were submitted to immunoprecipitation using anti-Trio or normal rabbit IgGs coupled to protein G-Sepharose beads. Immunoprecipitated proteins and 10% of the total cell lysates were submitted to SDS-PAGE, and the presence of DCC and Trio was detected by Western blotting using the appropriate antibodies. (C and D) Lysates of COS-7 cells transfected with pRK5 (EV), pRK5-DCC, pEGFP-Trio, pRK5-HA-Nck-1, or pRK5myc-PAK1, alone or as indicated, were incubated with GST and with GST-Nck (C) or GST-PAK (D). GST pull-down proteins (pull-down) and 10% of the total cell lysates were submitted to SDS-PAGE, and proteins were detected by Western blotting analysis using anti-DCC, anti-GFP, anti-PAK1, and anti-Nck-1 antibodies. (E) Lysates of COS-7 cells transfected with pEGFP-Trio(1-1813), pEGFP-Trio(1813-3038), pEGFP-Trio(1-1203), or pEGFP-Trio(1203-1813) were incubated with GST or GST-PAK. GST pull-down proteins (pull-down) and 8% of the total cell lysates were submitted to SDS-PAGE, and proteins were detected by Western blotting analysis using anti-GFP antibodies. IP, immunoprecipitation; TCL, total cell lysates.