Figure 1: Measurement of basal IRS1 protein stability in different cell lines. (a) Cells were treated for 6 hours with the following drugs prior to cell lysis: cycloheximide (40 μM), MG-132 (20 μM), MLN4924 (3 μM). Subsequently, Western blot analysis was performed. (b) HEK293T cells were pretreated with actinomycin D (5 μg/mL) for 25 min before adding MLN4924 (3 μM) for 4 hours, as indicated. (c) HEK293 cells were preincubated with MLN4924 (1 μM) for 2 hours before adding cycloheximide (40 μM) for the respective time points. Cell lysates were analyzed using Western blotting.