Figure 1.

The interaction between the COG complex and Sly1 is mediated by the Cog4 subunit. (A) An endogenous COG complex interacts with Sly1. HeLa cell lysate was subjected to immunoprecipitation (IP) with anti-Cog3, anti-Cog7 or anti-Cog4 specific antibodies. Preimmune (P.I.) sera of Cog3 and Cog4 were used as a control. The presence of Sly1 in the immunocomplexes of the indicated COG subunits was determined by immunobloting (IB) with anti-Sly1 antibody. The interaction between the different COG subunits was assessed by immunobloting with the indicated anti-Cog antibodies. (B) Sly1 interacts with endogenous COG subunits. HeLa cells were transiently transfected with expression vector encoding Myc-tagged Sly1. Sly1-Myc was immunoprecipitated by anti-Myc antibody and the presence of Cog3, Cog7 or Cog4 in Sly1 immunocomplexes was determined by immunoblotting with the indicated anti-Cog antibodies. (C) Cog4 interacts with recombinant GST-Sly1. HEK293 cells were transiently transfected with expression vectors encoding the eight different subunits of the COG complex as Myc-tagged proteins. The cell lysates were incubated with either GST or GST-Sly1 bound to glutathione-agarose beads. The samples were washed and then resolved by SDS–PAGE, transferred to a nitrocellulose membrane and immunoblotted with anti-Myc antibody (left panel). The expression level of each COG subunit was determined by western blotting with anti-Myc antibody (right panel). (D) Cog4-Myc interacts with Sly1-HA. HEK293 cells were either transiently transfected with an expression vector encoding Sly1-HA or cotransfected with Sly1-HA and the different Cog-Myc subunits. The COG subunits were immunoprecipitated with anti-Myc antibody, and their association with Sly1-HA was determined by immunoblotting with anti-HA antibody. The expression level of the transfected proteins was assessed by immunoblotting of total cell lysates with the indicated antibodies (lower panels).