FBXL14 Antibody - #DF13005

Figure 3. SIRT1 deacetylates and stabilizes β-TrCP1 protein to downregulate Snail1. (A) Caco2 cells stably expressing shRNAs against SIRT1 were subjected to Western blotting. (B) Caco2 cell lysates were subjected to immunoprecipitation (IP) with anti-β-TrCP1, using normal mouse or rabbit IgG as a control, followed by Western blot analyses. (C) Caco2 cells stably coexpressing β-TrCP1 and shRNAs against SIRT1 were subjected to Western blotting. (D) Caco2 cells treated with or without DSS were subjected to Western blotting. (E) HCT116 cells were treated with 4% DSS for 8 hours and the whole cell lysates were subjected to Western blotting. (F) Caco2 cells stably overexpressing SIRT1 were treated with 4% DSS for 8 hours. Whole-cell lysates were subjected to Western blotting. (G) Caco2 cells stably overexpressing β-TrCP1 were treated with 4% DSS for 8 hours. Whole-cell lysates were subjected to Western blotting. (H) Caco2 cells pretreated with 4% DSS for 8 hours or stably expressing shRNAs against SIRT1 were subjected to qPCR analyses for β-TrCP1. (I) To examine the effects of DSS on ubiquitination of β-TrCP1, 293FT cells were cotransfected with the relevant expression plasmids for 48 hours, followed by treatment with 4% DSS for 12 hours. Before harvesting, cells underwent treatment with 20 μM MG132 for 6 hours. Ubiquitination of β-TrCP1 was examined by denature-IP-western analyses. (J) The β-TrCP1 protein half-lives were measured by CHX assays in Caco2 cells pretreated with 4% DSS for 8 hours. (K and L) To examine the effects of SIRT1 on ubiquitination of β-TrCP1 and Snail1, 293FT cells were cotransfected with indicated expressing plasmids. Cells were treated with 20 μM MG132 for 6 hours before collection. Ubiquitination of Snail1 was examined by denature-IP-western analyses. (M) The β-TrCP1 and Snail1 protein half-lives were measured by CHX assays in Caco2 cells stably overexpressing SIRT1. (N and O) Caco2 cell lysates were subjected to IP with anti-SIRT1 (H) or anti-β-TrCP1 (I), using normal mouse or rabbit IgG as a control, followed by Western blot analyses. (P) HEK-293T cells cotransfected with His/Myc-β-TrCP1 and Flag-SIRT1 were subjected to IP and Western blotting analyses.