Fig. 3. Cornichon interacts with TGF family members. (A) CNIH interacts with TGF. Left: lysates of HeLa cells expressing TGF and/or Myc-tagged CNIH subjected to immunoprecipitation with anti-Myc, and immunoblotting with indicated antisera. Right: total cell lysates subjected to immunoblotting. TGF co-precitated with CNIH-Myc only when coexpressed and not when separately expressed and cell lysates were mixed (lane 5). CNIH did not interact with SEC61 (Sec61), an ER resident protein, or transferrin receptor (Tf-R). (B) CNIH interacts with amphiregulin (AR). Left: lysates of HeLa cells expressing Myc-tagged AR and/or Flag-tagged CNIH, subjected to immunoprecipitation with anti-Flag, and immunoblotting with anti-Myc. Right: total cell lysates subjected to immunoblotting to detect AR. Immature AR is marked. (C) Drosophila Cni interacts with Grk. S2 cells were transfected or not transfected to express Myc-tagged dCni (dCni-Myc) and treated or not with dsRNAi targeting dCni. Lysates were processed for Grk immunoprecipitation and immunoblotting with anti-Myc or anti-Grk antibodies. dCni-Myc co-precipitated with Grk, only when Grk and dCni-Myc were coexpressed in the absence of dsRNAi targeting Cni. dsRNAi targeting Cni did not affect Grk expression. (D) The EGF core of transmembrane TGF is required for interaction with CNIH. HeLa cells were transfected to express HA-tagged CNIH and full-length TGF, TGFC or TGFE. Cell lysates were subjected to anti-HA immunoprecipitation, and immunoblotting to detect CNIH-associated TGF. TGF and TGFC were detected using an anti-TGF antibody Ab-1, and TGFE was detected using anti-Myc. Lysates of non-transfected cells served as controls. Dividing vertical white lines indicate parts from the same gel (A,D).