Figure 2: Effect of Cullin-5 knockdown on basal IRS1 protein concentrations. (a) Negative control siRNA (NC) or two different Cullin-5 siRNAs were transfected into HEK293 cells, followed by transfection of Cul5-V5 after 24 hours. After further 24 hours, the cells were lysed with detergent lysis buffer and analyzed by Western blotting. (b) HEK293 cells were transfected with negative control or the respective Cul5 siRNA. After three days, the cells were treated with cycloheximide (40 μM) for 6 hours, lysed, and analyzed by Western blotting.