Figure 5.—

Co-immunoprecipitation assay to detect protein–protein interaction. (A) Co-immunoprecipitation of Dug2p and Dug3p. Tagged constructs of Dug2cMyc and Dug3HA were cotransformed or transformed along with vectors in ABC710. Cell extracts were prepared and immunoprecipation and immunoblotting were performed as described in materials and methods. Immunoprecipitation experiments were performed using anti-cMyc antibody (lanes 2, 4, and 5). The cell extracts of transformants as indicated were also loaded (lanes 1, 3, and 6). SDS–PAGE was followed with immunoblotting using anti-HA to detect Dug3HA. (B) Co-immunoprecipitation of Dug1p and Dug2p. Tagged constructs of Dug1His₈ and Dug2cMyc were cotransformed or transformed along with vectors in ABC710. Cell extracts were prepared and immunoprecipation and immunoblotting were performed as described in materials and methods. Immunoprecipitation experiments were performed using anti-His antibody (lanes 2 and 4). The cell extracts of transformants as indicated were also loaded (lanes 1, 3, and 5). SDS–PAGE was followed with immunoblotting using anti-cMyc to detect Dug2cMyc. (C) Co-immunoprecipitation of the Dug1p homodimer. Tagged constructs of Dug1His₈ and Dug1HA were cotransformed or transformed along with vectors in ABC710. Cell extracts were prepared and immunoprecipation and immunoblotting were performed as described in materials and methods. Immunoprecipitation experiments were performed using anti-His antibody (lanes 1 and 5). The cell extracts of transformants as indicated were also loaded (lanes 2, 3, and 6). SDS–PAGE was followed with immunoblotting using anti-HA to detect Dug1HA.