Fig. 7: SART3 facilitates BARD1 deubiquitination via enhancing BARD1-USP15 association.

a HEK293T cells transfected with the indicated constructs were treated with CPT (5 µM, 2 h), followed by immunoprecipitation with anti-GFP agarose beads and immunoblotting with the indicated antibodies. Asterisks indicate non-specific bands. b U2OS cells transfected with siRNAs were treated with CPT and recovered for 0 h, 2 h, or 5 h, respectively. Triton-insoluble fractions (TIF) and whole-cell lysates (WCL) were harvested for immunoblotting. “R” stands for recovery. c, d HEK293T cell expressing GFP-SART3 and Myc-BARD1 were immunoprecipitated with anti-GFP beads, followed by immunoblotting with anti-Myc or anti-GFP (c). HEK293T cell lysates were immunoprecipitated with anti-BARD1 antibodies, followed by immunoblotting with antibodies against SART3 or USP15 (d). e Schematic representation of the SART3 truncated mutants (top). HEK293T cells co-transfected with Myc-BARD1 and GFP-SART3 truncations were harvested for Co-IP with anti-GFP beads, followed by immunoblotting with antibodies against Myc, GFP, or USP15. f HEK293T cells were co-transfected with GFP-USP15, Myc-BARD1, and Flag-SART3-WT or truncations. 36 h later, cells were treated with CPT, followed by Co-IP. g HEK293T cells transfected with the indicated siRNAs were co-transfected with HA-Ub, Flag-BARD1 and GFP-SART3-WT or truncations. 36 h later, cells were treated with CPT and subjected to denatured IP with anti-Flag agarose beads, followed by immunoblotting with the indicated antibodies. h, i U2OS cells transfected with siNC, siSART3, siUSP15-1, or both siSART3 and siUSP15-1 were treated with CPT (5 µM, 2 h) and further cultured for 2 h, followed by immunofluorescence with antibodies against BRCA1 or BARD1. Representative images are shown (left). Proportion of cells with BRCA1 or BARD1 foci was measured (right). Scale bars for overall and magnified images are respectively 50 and 10 µm. j U2OS-DR-GFP cells stably expressing Flag, Flag-SART3, Flag-SART3-ΔHAT4-7 or Flag-SART3-ΔRRM were transfected with either siNC or siSART3. After 24 h, cells were infected with I-SceI lentivirus. The percentage of GFP-positive cells was quantitated by FACS 48 h post I-SceI lentivirus infection. In (h–j), error bars represent mean ± SEM (N = 3 independent experiments), and p values were calculated using unpaired two-tailed Student’s t test. Source data are provided as a Source Data file.