Fig. 7. AS160 interacts with Rip11. (A) CHO.T cells were transfected with plasmids encoding FLAG-tagged wild-type AS160 (AS160-WT), along with either Xpress-tagged wild-type Rip11 (Rip11-WT) or Rip11-I630E, or the Xpress vector, as indicated. The cells were incubated in the absence or presence of 100 nM insulin for 10 minutes, as shown, before lysis. Cell lysates were blotted with antibodies against Xpress (lysate: IB Rip11) or against FLAG (lysate: IB AS160) to assess the level of expression of these proteins. Alternatively, Rip11 complexes were immunoprecipitated using the antibody against the Xpress tag, and these complexes were blotted with antibodies against Xpress (IP Rip11: IB Rip11) or FLAG (IP Rip11: IB AS160) to detect the presence of Xpress-tagged Rip11 and FLAG-tagged AS160 in the precipitates, respectively. (B) The extent of the interaction of the wild-type (WT) and I630E mutant Rip11 with AS160 was quantitated by densitometry and is plotted as the mean ± s.e.m. for four separate preparations of adipocytes.