Figure 2.

CHIP Regulates DAF-2/INSR Protein Level

(A, B) chn-1(by155) shows increased levels of DAF-2 protein, analyzed by immunoblotting of worm lysates of indicated age (day 0; L4 worms) (mean values were obtained in n = 6 independent experiments).

(C) Lack of UFD-2 does not stabilize DAF-2.

(D) chn-1(by155) mutation does not increase the global level of ubiquitylated proteins in young or aged adult worms.

(E) Ubiquitous depletion of dCHIP increases levels of dINSR (detected in whole fly extracts) (mean values were obtained in n = 5 independent experiments).

(F) dINSR stabilized upon dCHIP depletion does not accumulate in detergent-insoluble aggregates. Fly heads were lysed in SDS-containing buffer and the resultant extract was separated in an SDS-soluble and -insoluble fraction by centrifugation at 100,000 × g.

(G) Depletion of CHIP in human HEK293 cells causes increased levels of INSR as determined by western blot, whereas mRNA levels remain unchanged (mean values were obtained in n = 5 independent experiments).

(H) Overexpression of CHIP results in decreased level of INSR in HEK293 cells (mean values were obtained in n = 5 independent experiments).

(B, E, G, and H) Data are means ± SEM. ^∗p < 0.05, ^∗∗p < 0.01.

See also Figure S2.