Figure 2

CB, gephyrin and eIF3H interaction in a mammalian cell context. 293T cells were cotransfected with plasmids enconding the indicated combinations of the epitope-tagged proteins: (A) Myc-CB (~62 kDa) and Flag-gephyrin (~95 kDa), Flag-CB (~62 kDa) and Myc-gephyrin (~95 kDa); (B) Flag-CB and Myc-eIF3H (~40 kDa), Myc-eIF3H and Flag-gephyrin, Myc-CB and Myc-eIF3H and Flag-gephyrin. As negative controls 293T cells were cotransfected with a plasmid encoding the Flag-tag alone along with plasmids encoding the Myc-tagged proteins (data not shown). Immunoprecipitations were carried out using anti-Flag antibody and immunoblots were performed using anti-Myc antibody (top panels). Coimmunoprecipitations of CB-gephyrin (A, top panels), CB-eIF3H (B, top left panel), gephyrin-eIF3H (B, top middle panel), CB-gephyrin-eIF3H (B, top right panel) were observed. In analyzing negative controls, no detectable protein coimmunoprecipitation was accomplished (data not shown). Expression levels of the ectopic expressed proteins were confirmed by direct immunoblotting of equivalent amounts of the lysates using anti-Flag and/or anti-Myc antibodies as shown in the lower panels. All results shown are representative of two-four independent experiments. IP = antibody used for immunoprecipitation; Blot = antibody used for immunoblot analysis; IgG-Hc = immunoglobulin heavy chain (~55 kDa).