Figure 4: Role of the Cullin-5 E3 ligase in TPA-induced IRS1 degradation. (a) HEK293 cells were treated TPA (10 nM) for the last 24 hours, with MLN4924 (3 μM) (24 hours) and MG-132 (20 μM) (6 hours), as indicated. (b) HEK293 cells were transfected with negative control or Cullin-5 siRNA. After two days, TPA (10 nM) was added for 24 hours. Subsequently, the cells were lysed for Western blot analysis. (c) HEK293T cells were transfected with the respective amounts of SOCS plasmid as indicated. Immunoblot analysis of lysates was subsequently carried out.