FIGURE 5.

AS160(1–532) interacts with full-length AS160, forms heterodimers, and inhibits 14-3-3 binding to AS160 in HEK293 cells. HEK293 cells were cotransfected with FLAG-tagged full-length AS160 and HA-AS160(1–532). After 18 h of expression, cells were serum-starved, left unstimulated (basal (B)) or stimulated with 50 ng/ml IGF1, and then harvested and lysed in detergent. FLAG-AS160 or HA-AS160(1–532) was immunoprecipitated (IP) with anti-FLAG or anti-HA tag antibody, respectively. The immunoprecipitated proteins were subjected to SDS-PAGE and immunoblotted with anti-FLAG, anti-HA, anti-pan-14-3-3, or anti-PAS antibody. The relative levels of 14-3-3 (A) and FLAG-tagged phosphorylated AS160 (pAS160; B) pulled down were quantified and normalized to the level of expression of FLAG-tagged full-length AS160. Results are means ± S.E. from three independent experiments. *, p < 0.05 (comparison of FLAG-AS160 alone versus FLAG-AS160 plus HA-AS160(1–532)). C, representative immunoblots.