Fig. 7. Expression of E4B and CHIP accelerated the degradation of the identified substrates.

(A) E4B reduced the steady-state levels of the substrate proteins in the HEK293 cells. Cells were transfected with an increasing amount of E4B plasmid. Levels of the E4B substrates were assayed with immunoblots of the cell lysate probed with substrate-specific antibodies. Approximately 5 × 10⁶ cells were used for each transfection of the E4B plasmid. (B) CHIP reduced the steady-state levels of the substrate proteins in the HEK293 cells. Assays were performed with a similar protocol to (A). (C and D) Quantitative analysis of substrate levels in correlation with E4B and CHIP expression, respectively. Intensity of the bands in (A) and (B) was plotted against the amount of pLenti E4B or CHIP plasmid used for transfection, assuming 100% substrate protein when an empty plasmid was used for mock transfection. Results were the average of three repeats.