The Role of the Cullin-5 E3 Ubiquitin Ligase in the Regulation of Insulin Receptor Substrate-1

Figure 3

Effect of mTOR/S6K1 and TPA on IRS1 protein stability. (a) HEK293T cells were treated with rapamycin (20 nM) and cycloheximide (40 μM), as indicated, for 6 h followed by cell lysis and Western blot analysis. IRS1 protein abundance was determined by densitometry analysis indicated above the band as fold compared to the untreated control. All forms of IRS1 (shifted and not shifted) were included in the densitometry analysis. (b) HEK293T cells were preincubated with Gö8963 (5 nM) for one hour before 24 hour incubation with TPA (10 nM), as indicated. (c) HEK293 cells were pretreated with 10 nM TPA for 1 hour before cycloheximide chase was conducted using the indicated time points. Immunoblot analysis of lysates was then carried out. (d) HEK293T cells were transfected with the specified amounts of IRS1. After the cells reached subconfluence, TPA (10 nM) was added to the medium for 24 hours before cell lysis. (e) HEK293 cells were treated with TPA (10 nM) for 24 hours followed by Western blot analysis of cell lysates for endogenous β-catenin. (f) HEK293 cells were transfected with 0.5 μg of the indicated β-catenin plasmids. After two days, TPA (10 nM) was added for 24 hours followed by Western blot analysis of cell lysates with the indicated antibodies.