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. 2018 Sep 1;8(9):1823–1836.

Figure 3.

Figure 3

USP2a stabilizes β-catenin. (A) Top panel: HEK293T cells were co-transfected with MYC-β-catenin, MYC-GFP, and SFB-tagged GFP or USP2a, treated with 100 µg/ml cycloheximide (CHX), harvested at different time points, and then immunoblotted with antibodies against MYC, β-actin, and FLAG. MYC-GFP serves as the control for transfection. Bottom panel: quantification of β-catenin protein levels (normalized to MYC-GFP). (B) Top panel: MDA-MB-231 cells were transfected with the control vector (pLOC-RFP) or USP2a (pLOC-USP2a), treated with 100 µg/ml cycloheximide (CHX), harvested at different time points, and then immunoblotted with antibodies against USP2a, β-catenin, and CypB. Bottom panel: quantification of β-catenin protein levels (normalized to CypB). (C) Top panel: BT549 cells were transfected with USP2 shRNA (#1 or #2), treated with 100 µg/ ml CHX, harvested at different time points, and then immunoblotted with antibodies against USP2, β-catenin, and HSP90. Bottom panel: quantification of β-catenin protein levels (normalized to HSP90). (D and E) HEK293T cells were co-transfected with HA-ubiquitin, MYC-β-catenin, and SFB-tagged GFP (D) or USP2a (E). Cells were pretreated with ML364 (0, 3, 10, or 30 µM) for 4 hours and then treated with 10 µM MG132 in the presence of ML364 for 6 hours. β-catenin was immunoprecipitated with anti-MYC beads, followed by immunoblotting with antibodies against HA and MYC. (F) HEK293T cells were transfected with HA-ubiquitin, pretreated with 0, 3, or 10 µM ML364 for 24 hours, and then treated with 10 µM MG132 in the presence of ML364 for 6 hours. Polyubiquitinated proteins were immunoprecipitated with anti-HA beads, followed by immunoblotting with antibodies against β-catenin and HA. (G) HEK293T cells were co-transfected with SFB-USP2a and MYC-β-catenin and treated with ML364 (0, 3, or 10 µM) for 24 hours, followed by pulldown with S-protein beads and immunoblotting with antibodies against MYC and FLAG. (H) Immunoblotting of β-catenin, USP2a, and CypB in BT549, LM2, and HCC1806 cells treated with ML364 at the indicated doses.