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. 2024 Jan 17;14(4):1624–1643. doi: 10.1016/j.apsb.2024.01.006

Figure 3.

Figure 3

USP48 deubiquitinates HMGA2. (A) HEK293T cells were co-transfected with Myc-HMGA2, His-Ub, and either HA-tagged USP48-WT or USP48-C98S. Cell lysates were then subjected to IP using anti-Myc magnetic beads followed by IB analysis with antibodies against His, HA, and Myc. Prior to harvesting, the cells were treated with 10 μmol/L MG132 for 6 h. (B) Increasing amounts of HA-tagged USP48 and equal amounts of Myc-HMGA2 were transfected into the cells, followed by IP with Myc antibodies and IB analysis with anti-His antibodies to detect ubiquitinated HMGA2 after treatment with MG132 for 6 h. (C) DLD1 cells were transfected with USP48-WT or USP48-C98S. After 48 h, the cells were treated with MG132 for 6 h. The whole-cell lysates were subjected to IP with HMGA2 antibodies and IB analysis with anti-Ub antibodies to detect ubiquitinated HMGA2. (D) SW480 cells were transfected with shRNA as indicated, and lysates were subjected to IP using HMGA2 antibody, followed by IB analysis with antibodies against Ub and HMGA2. (E) HCT116 cells were transfected with shRNA as indicated, and lysates were subjected to IP using HMGA2 antibody, followed by IB analysis with antibodies against Ub and HMGA2. Cells were treated with 10 μmol/L MG132 for 6 h prior to harvesting. Before IP, lysates were denatured at 95 °C for 5 min in the presence of 1% SDS, then diluted tenfold with lysis buffer and sonicated. (F) The ubiquitinated Myc-HMGA2 protein was subjected to in vitro deubiquitination assay using USP48-WT or USP48-C98S, and the reaction mixes were analyzed by IB analysis. (G) Myc-HMGA2 was co-transfected with either USP48-WT or USP48 deletion mutants into HEK293T cells. Whole-cell lysates were subjected to IP using Myc antibody, followed by IB analysis with anti-His antibodies after treatment with MG132 for 6 h to detect ubiquitinated HMGA2. (H) De-ubiquitination assay of HMGA2 WT and mutant domains (D1‒D4) was performed in HEK293T cells co-transfected with His-Ub and HA-USP48, followed by treatment with 10 μmol/L MG132 for 6 h. (I) De-ubiquitination assay of HMGA2 in HEK293T cells co-transfected with His-Ub, HA-USP48, Myc-HMGA2-K26R, and Myc-HMGA2-K34R and treated with 10 μmol/L MG132 for 6 h. (J) De-ubiquitination assay of HMGA2 in HEK293T cells co-transfected with His-Ub, HA-USP48, Myc-HMGA2-K26R and treated with 10 μmol/L MG132 for 6 h. (K) Myc-HMGA2, HA-USP48 and His-Ub plasmids with different ubiquitin linkages (K6, K11, K27, K29, K33, K48 or K63) were co-transfected into HEK293T cells to analyze HMGA2 ubiquitination. The data shown in all panels are representative of three independent experiments.