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. 2021 Mar 4;11(9):4421–4435. doi: 10.7150/thno.53901

Figure 2.

Figure 2

CK1δ/ε interacts with AES and regulates AES stability. (A) HEK293T cells were transfected with AES-V5 plasmid along with expression vectors for Flag-CK1α, Flag-CK1δ, Flag-CK1ε, and Flag-CK1γ1, respectively. Cell lysates were immunoprecipitated with anti-Flag M2 beads. The interaction of AES with different CK1 isoforms was detected by immunoblot analysis. The asterisk represents the IgG heavy chain. (B) HEK293T cells were transfected with AES-V5 plasmid together with expression vectors for Flag-CK1δ and Flag-CK1ε, respectively. Cell lysates were immunoprecipitated with anti-V5 agarose beads. The interaction of AES with CK1δ or CK1ε was visualized by immunoblotting. (C) Cell lysates from HCT116 cells were subjected to IP with IgG or anti-AES antibody. The interaction of AES with CK1δ or CK1ε was visualized by immunoblotting. (D) HCT116 cells were infected with shC (control), shCK1δ-1/shCK1ε-1 mixture (shCK1-1), or shCK1δ-2/shCK1ε-2 mixture (shCK1-2) lentivirus, then cell lysates were subjected to immunoblotting with the indicated antibodies. (E) HCT116 cells were infected with shC, shCK1δ-1/shCK1ε-1 mixture (shCK1-1), or shCK1δ-2/shCK1ε-2 mixture (shCK1-2) lentivirus and treated with 100 μg/ml CHX for the indicated periods of time. The expression of AES was detected by immunoblotting. The protein level of AES was quantitated by densitometry and normalized to GAPDH. (F) HCT116 cells were treated with 10 μM MG132 for 6 h, and the protein level of AES was detected by immunoblotting. (G) HCT116 cells were infected with shC, shCK1δ-1/shCK1ε-1 mixture (shCK1-1), or shCK1δ-2/shCK1ε-2 mixture (shCK1-2) lentivirus and treated with or without 10 μM MG132 for 6 h. Cell lysates were immunoprecipitated with AES antibody. The levels of AES-Ub and AES were detected by immunoblotting. (H) HCT116 or HT29 cells were treated with or without 0.25 μM IC261 for 24 h and 10 μM MG132 for 6 h before harvesting. Cell lysates were subjected to IP with AES antibody. The levels of AES-Ub and AES were detected by immunoblotting. (I) HCT116 cells were treated with DMSO or the indicated amounts of IC261 for 24 h. Cell lysates were subjected to immunoblotting with the indicated antibodies. (J) HCT116 cells were treated with DMSO, 2 μM PF670462 or 100 nM SR3029 for 24 h, then cell lysates were subjected to immunoblotting with the indicated antibodies. Values are shown as means ± SD (n = 3). *P < 0.05; Two-way ANOVA.