Fig. 4: BAP1 N-Terminal domain and catalytic activity are critical for pVHL stabilization and tumor suppression in PDAC.

A Schematic of BAP1 constructs: WT (wild-type), M1 (1–240 amino acids), M2 (241–594 amino acids), and M3 (595-C terminal) constructs. pIRES vector, pIRES-BAP1 WT or truncation mutants were transfected in HEK293T cells. Cell lysates were subjected to S-protein agarose pulldown, and the interactions with pVHL were detected by western blot. Data are representative of three independent experiments. B PANC-1 cells stably expressing BAP1 shRNA were transfected with vector, BAP1 WT, the catalytic-inactive mutant C91S, or the Δ1–240 truncation mutant (lacking the pVHL interaction region). Western blotting was performed with the indicated antibodies. Data are representative of three independent experiments. C Cells stably expressing BAP1 shRNA were transfected as indicated. Cell lysates were subjected to immunoprecipitation with anti-HA magnetic beads, and pVHL ubiquitination was assessed by western blotting with anti-ubiquitin antibody. Data are representative of three independent experiments. D Cell proliferation assay in PANC-1 cells as in (B). Results represent the mean ± s.d. of three independent experiments (biological replicates). Statistical significance was determined by one-way ANOVA followed by Tukey’s multiple comparisons test. E PANC-1 cells as in (B) were treated with indicated concentrations of gemcitabine or oxaliplatin, and cell viability was measured. Results represent the mean ± s.d. of three independent experiments (biological replicates). Statistical significance was determined by one-way ANOVA followed by Tukey’s multiple comparisons test. F Cells stably expressing BAP1 shRNA were transfected with vector, BAP1 WT, or cancer-related mutants. Western blotting was performed with the indicated antibodies. Data are representative of three independent experiments. G Cells stably expressing BAP1 shRNA were transfected with vector, BAP1 WT or cancer-related mutants. Cell lysates were subjected to immunoprecipitation with anti-FLAG antibody, and western blotting was performed with the indicated antibodies. Data are representative of three independent experiments. H Cells stably expressing BAP1 shRNA were transfected with indicated plasmids. Cell lysates were subjected to immunoprecipitation with anti-HA magnetic beads, and pVHL ubiquitination was measured by western blotting. Data are representative of three independent experiments. I Cell proliferation assay was performed in PANC-1 cells as in (F). Results represent the mean ± s.d. of three independent experiments (biological replicates). Statistical significance was determined by one-way ANOVA followed by Tukey’s multiple comparisons test. J PANC-1 cells as in (F) were treated with indicated concentrations of gemcitabine or oxaliplatin, and cell viability was measured. Results represent the mean ± s.d. of three independent experiments (biological replicates). Statistical significance was determined by one-way ANOVA followed by Tukey’s multiple comparisons test.