FIGURE 4.

Homo-oligomerization of RGS7. A, HEK293T cells were co-transfected with HA-RGS7 and Gβ₅, FLAG-RGS7 and Gβ₅, or all the three constructs. Cell lysates were subjected to IP using anti-FLAG antibody, as described under “Experimental Procedures.” The eluates from the resin were analyzed using anti-FLAG, HA, and Gβ₅ antibodies. B, reciprocal IP using anti-HA beads. Cell transfection and lysis were done as in A; immunoprecipitation was performed using anti-HA antibodies, and the bound material was probed for the presence of FLAG-tagged RGS7. C, mCherry-RGS7 was co-immunoprecipitated with YFP-RGS7 from N2A cell lysates. The experiment was performed essentially as in A or B. D, two separate cell cultures were transfected with either FLAG-RGS7/Gβ₅ or HA-RGS7/Gβ₅, harvested, and lysed. The two lysates were mixed (mix) prior to co-IP with immobilized anti-FLAG antibody. In parallel, FLAG-RGS7, HA-RGS7, and Gβ₅ plasmids were co-transfected (co-trans), and cell lysates were analyzed by co-IP and immunoblot.