Figure 3.

SKP2 interacts with AES and mediates its degradation. (A) HEK293T cells were transfected with AES-V5 plasmid along with expression vector for Flag-SKP2, then cells were lysed and subjected to IP with anti-Flag M2 beads. Immunoblot analysis was performed with the indicated antibodies. (B) Similar to (A) except anti-V5 agarose beads were used in Co-IP. (C) Cell lysates from HCT116 cells were subjected to IP with IgG or anti-AES antibody. Immunoblot analysis was performed with the indicated antibodies. (D) HEK293T cells were transfected with the indicated expression plasmids and treated with or without 10 μM MG132 for 6 h before harvesting. Cell lysates were subjected to immunoblotting with the indicated antibodies. The plasmid pEGFP-N1 was used to monitor transfection efficiency. (E) HEK293T cells were transfected with the indicated expression plasmids and treated with or without 10 μM MG132 for 6 h before harvesting. Cell lysates were immunoprecipitated with anti-V5 agarose beads. The levels of AES-Ub and AES were detected by immunoblotting. (F) HCT116 cells were infected with shC, shSKP2-1, or shSKP2-2 lentivirus, then cell lysates were subjected to immunoblotting with the indicated antibodies. (G) HCT116 cells were infected with shC, shSKP2-1 or shSKP2-2 lentivirus and treated with or without 10 μM MG132 for 6 h, then cell lysates were immunoprecipitated with AES antibody. The levels of AES-Ub and AES were detected by immunoblotting. (H) HCT116 cells were infected with shC, shSKP2-1, or shSKP2-2 lentivirus and treated with 100 μg/ml CHX for the indicated times. The expression of AES was detected by immunoblotting. The protein level of AES was quantitated by densitometry and normalized to GAPDH. Values are shown as means ± SD (n = 3). *P < 0.05; Two-way ANOVA.