Fig 2: Fbxo45 negatively regulates USP49 stability through ubiquitinated degradation.A IB analysis of whole-cell lysates (WCLs) derived from Panc-1 cells transfected with the indicated plasmids, which were treated with 10 µM MG132 for 6 h before harvesting. B IB analysis of WCLs derived from Panc-1 cells transfected with Fbxo45 constructs or EV. EV: empty vector. C IB analysis of WCLs derived from Panc-1 cells transfected with Fbxo45 siRNA or the negative control (NC). D qRT-PCR analysis to detect Fbxo45 and USP49 mRNA levels after Fbxo45 depletion in 293 T cells. Data are shown as the mean ± SD of three independent experiments. ***p < 0.001 compared to NC. E IB analysis of WCLs derived from Panc-1 cells after the specified duration of 100 µg/ml cycloheximide (CHX) transfection with Fbxo45 siRNA. F USP49 protein abundance in (E) was quantified and plotted. G IB analysis of immunoprecipitates (IPs) and WCLs derived from Panc-1 cells transfected with the indicated plasmids. Cells were treated with 10 µM MG132 for 6 h before harvesting. H IB analysis of ubiquitination products and WCLs derived from Panc-1 cells transfected with the indicated constructs. Cells were treated with 10 µM MG132 for 6 h before harvesting.

Fig 3: Fbxo45 promotes cell growth, migration, and invasion in PC cells.A MTT assays to detect cell proliferation in Panc-1 cells transfected with Fbxo45 siRNAs (Right) or Fbxo45 constructs (Left). Data are shown as mean ± SD of three independent experiments. ***p < 0.001 compared to control. B, C Tunel assays to detect cell apoptosis of Panc-1 cells transfected with Fbxo45 constructs (B) or Fbxo45 siRNAs (C). ***p < 0.001 compared to control. D Wound healing assays to analyze cell migration capacity of Panc-1 cells transfected with Fbxo45 constructs. **p < 0.01 compared to control. E Transwell assays to analyze cell migration and invasion capacity of Panc-1 cells transfected with Fbxo45 cDNA. **p < 0.01 compared to control, ***p < 0.001 compared to control. F Transwell assays to analyze cell migration and invasion capacity of Panc-1 cells transfected Fbxo45 siRNAs. **p < 0.01 compared to control, ***p < 0.001 compared to control.

Fig 4: Fbxo45-induced ubiquitination and degradation of USP49 depends on its SPRY domain.A Schematic of Fbxo45 domains and its mutant construct. B IB analysis of WCLs derived from Panc-1 cells transfected with the indicated plasmids. C IB analysis of WCLs derived from Panc-1 cells after the specified duration of 100 μg/ml cycloheximide (CHX) transfection with the indicated constructs. D USP49 protein abundance in (C) was quantified and plotted. E IB analysis of IPs and WCLs derived from Panc-1 cells transfected with the indicated plasmids. Cells were treated with 10 μM MG132 for 6 h before harvesting. F IB analysis of ubiquitination products and WCLs derived from Panc-1 cells transfected with the indicated constructs. Cells were treated with 10 μM MG132 for 6 h before harvesting.

Fig 5: Fbxo45 binds to a conserved motif in USP49 and NEK6 enhances the degradation of USP49.A Schematic of USP49 domains and its mutant construct. B IB analysis of WCLs derived from Panc-1 cells transfected with the indicated plasmids. C IB analysis of IPs and WCLs derived from Panc-1 cells transfected with the indicated constructs. Cells were treated with 10 μM MG132 for 6 h before harvesting. D IB analysis of ubiquitination products and WCLs derived from Panc-1 cells transfected with the indicated constructs. Cells were treated with 10 μM MG132 for 6 h before harvesting. E IB analysis of WCLs derived from Panc-1 cells transfected with NEK6 siRNAs. F IB analysis of WCLs derived from Panc-1 cells transfected with the indicated constructs. G IB analysis of ubiquitination products and WCLs derived from Panc-1 cells transfected with the indicated constructs. Cells were treated with 10 μM MG132 for 6 h before harvesting. H IB analysis of ubiquitination products and WCLs derived from Panc-1 cells transfected with the indicated constructs. Cells were treated with 10 μM MG132 for 6 h before harvesting.