Figure 3. COG6 interacts with a subset of intra-Golgi SNARE proteins.
(A) HeLa cells were transiently cotransfected with plasmids encoding fluorescent tagged SNAREs and COG6-myc. 24 h post-transfection, cells were collected, lysed, and GFP-SNARE interacting proteins were precipitated with anti-GFP antibodies. Immuno-precipitates, along with 10% of total input, were separated on a 12% SDS-PAGE gel, transferred to a nitrocellulose membrane, and probed with antibodies against myc (upper panel) and GFP (lower panel). Band densitometry was evaluated using Odyssey software. Co-immunoprecipitation efficiency values were calculated by dividing Co-IP by input. (B) COG6-myc demonstrated its strongest interaction with CFP-SNAP29 (8%) and CFP-GS27 (6%). (C) NRK cells were lysed and COG-interacting proteins were immunoprecipitated with affinity-purified anti-COG3, COG4 and COG6 IgGs. Immuno-precipitates, along with 10% of total input and flow through, were separated on a 12% SDS-PAGE gel, transferred to a nitrocellulose membrane, and probed with antibodies against GS27, COG3, COG4, and COG6.