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

Figure 1.

Figure 1

USP48 interacts with HMGA2. (A) HMGA2-interacting proteins were detected by mass spectrometry using anti-Myc affinity magnetic beads, following transfection of Myc-HMGA2 into HCT116 cells for 36 h. (B–C) IP assays were performed on HCT116 and SW480 cell lysates using control IgG, anti-USP48, or anti-HMGA2 antibodies, followed by detection of resulting immunoprecipitates with appropriate antibodies. (D) HEK293T and DLD1 cells were transfected with Myc-HMGA2 alone or in combination with HA-tagged USP48-WT or USP48 CS, and cell lysates were analyzed by IP with anti-HA affinity magnetic beads followed by IB with antibodies against Myc and HA. (E) Purified HA-USP48 or HA-USP48 C98S from HEK293T cells was incubated with purified recombinant GST-HMGA2 or GST, respectively. The retained USP48 or USP48 C98S on sepharose was detected using the HA antibody. (F) HCT116, DLD1 and SW480 cells were stained with USP48 antibody (red) and HMGA2 antibody (green), then analyzed using confocal microscopy. Nuclei were counterstained with DAPI (blue). Scale bar, 10 μm. (G) In situ PLA was used to detect the interaction between endogenous USP48 and HMGA2 in HCT116 and DLD1 cells. Representative merged PLA and nuclei (DAPI) images from the experiments are shown, with upper panel scaled at 10 μm and lower panel at 5 μm. (H) HEK293T cells were co-transfected with Myc-HMGA2 and either full-length HA-tagged USP48 or its deletion mutants, followed by IP using anti-HA magnetic beads and IB with antibodies against HA and Myc. (I) HEK293T cells were co-transfected with Myc-HMGA2 and either full-length HA-tagged USP48 or its deletion mutants, followed by IP using anti-Myc magnetic beads and IB with antibodies against HA and Myc. (J) In situ PLA was employed to investigate the interaction between exogenous Myc-HMGA2 WT and HA-USP48 △USP in HEK293T cells, with a scale bar of 10 μm. (K) In situ PLA was employed to investigate the interaction between exogenous HA-USP48 WT and Myc-HMGA2 D1 in HEK293T cells, with a scale bar of 10 μm. (L) HEK293T cells were co-transfected with HA-USP48 and either full-length Myc-tagged HMGA2 or its deletion mutants, followed by IP using anti-Myc magnetic beads and IB with antibodies against HA and Myc. (M) HEK293T cells were co-transfected with HA-USP48 and either full-length Myc-tagged HMGA2 or its deletion mutants, followed by IP using anti-HA magnetic beads and IB with antibodies against HA and Myc. The presented panels depict representative outcomes from three independent experiments. The panels presented illustrate representative results obtained from three independent experiments.