Figure 4.

CK1δ/ε is required for the interaction of SKP2 with AES and SKP2-mediated AES degradation. (A) HEK293T cells were transfected with the indicated expression plasmids and cell lysates were treated with or without λ-PPase before immunoprecipitating with anti-Flag M2 beads. Immunoblot analysis was performed with the indicated antibodies. The asterisk represents the IgG heavy chain. (B) The lysates from HCT116 and HT29 cells were treated with or without λ-PPase before immunoprecipitating with anti-AES antibody. Immunoblot analysis was performed with the indicated antibodies. (C) HEK293T cells were infected with shC, shCK1δ/ε-1, or shCK1δ/ε-2 lentivirus, followed by transfection with the indicated plasmids. Cell lysates were immunoprecipitated with anti-Flag M2 beads. The interaction of AES with SKP2 was detected by immunoblotting. (D) HEK293T cells were transfected with the indicated plasmids followed by treatment with DMSO, 0.25 μM IC261, or 2 μM PF670462 for 24 h. Cell lysates were subjected to IP with anti-Flag M2 beads. Immunoblot analysis was performed to detect the interaction between AES and SKP2. (E) HEK293T cells were transfected with the indicated plasmids, and then cell lysates were subjected to IP with anti-Flag M2 beads. Immunoblot analysis was used to detect the interaction between AES and SKP2 in the absence or presence of dominant negative mutant of CK1δ or CK1ε. Anti-V5 antibody was used to detect AES-V5 and CK1-V5. (F) HEK293T cells were transfected with the indicated plasmids and treated with or without 10 μM MG132 for 6 h. Cells lysates were subjected to immunoblotting with the indicated antibodies. Anti-V5 antibody was used to detect AES-V5 and CK1-V5.