Plasmids, cell culture and transfection

pcDNA3.1-HA-MIB1^wild-type was constructed by introducing HA epitope sequence and full-length human MIB1 gene coding region ({"type":"entrez-nucleotide","attrs":{"text":"NM_020774.3","term_id":"514825576","term_text":"NM_020774.3"}}NM_020774.3) into EcoRI/XhoI sites of pcDNA3.1 (ThermoFisher Scientific, #V79020). For pcDNA3.1-HA-MIB1^mutants, site-directed mutagenesis was carried out with QuikChange Lightning Site-Directed Mutagenesis Kit (Agilent, #210518) according to manufacturer’s instruction. Full-length MIB1 wild-type or mutants without stop codon were subcloned into SgfI/MluI sites of pCMV6-AC-GFP (Origene, #PS100010) to generate pCMV6-AC-GFP-MIB1^{wild-type or mutants}. For pCMV6-AC-GFP-MIB1^{p.T312K fs*55}, sequence after premature stop codon (including stop codon) was removed for C-terminal tGFP tag fusion. MIB1 mib repeat domain regions (aa 216-462) of wild-type, p.Q237H and p.W271G were inserted into SgfI/MluI sites of pCMV6-AN-Myc (Origene, #PS100012) to obtain pCMV6-AN-Myc-mib reps^{wild-type or p.Q237H or p.W271G}. Myc-Flag-tagged full-length human JAG1 (pCMV6-Entry-JAG1) was purchased from Origene (#RC210516). pCMV6-AN-DDK-JICD was generated using JAG1 intracellular domain (aa 1092-1218) subcloned into SgfI/MluI sites of pCMV6-AN-DDK (Origene, #PS100014). TP-1 Luciferase was generated by subcloning synthetic hexamer of Epstein-Barr virus (EBV) terminal protein 1 (TP1) gene promoter RBP-Jκ binding sites into KpnI/XhoI sites of pGL3-Basic [23]. pCBFRE Luciferase was purchased from Addgene (plasmid #26897) [24]. Renilla Luciferase reporter pGL4.74[hRluc/TK] was purchased from Promega (#E6921). HA-tagged tetrameric ubiquitin expression plasmid (HA-4 Ub) was kindly provided by Dr. Shimin Zhao at Fudan University.

HEK 293T cells were cultured in high-glucose Dulbecco’s Modified Eagle Medium (ThermoFisher Scientific, #11995065) supplemented with 10% FBS at 37°C with 5% CO₂. Cells were seeded and maintained overnight to reach 80% confluence at the time of transfection. Lipofectamine 3000 Transfection Reagent (ThermoFisher Scientific, #L3000015) was used for transient transfection following manufacturer’s protocol.