Fig. 8. DTL directly binds to SLTM and mediates its ubiquitination-dependent degradation.

A, B Co-immunoprecipitation (Co-IP) and western blot (WB) were performed to assess the interaction between exogenous DTL and SLTM in Huh-7 cells. C 2xHA-SLTM was transfected into 293T cells with or without 3xFlag-DTL. The effect of DTL on SLTM ubiquitination was analyzed by IP and WB. D WB analysis of 293T cells with DTL knockdown and those overexpressing DTL, both treated with cycloheximide (CHX, 50 mg/ml) for various time points to evaluate protein degradation. E Co-IP was employed to determine the binding domain of DTL that interacts with SLTM. F Schematic overview of the full-length DTL and its truncated mutants, depicted in plasmid constructs. G, H Quantitative real-time PCR (qRT-PCR) and WB were conducted to assess the expression levels of cleaved Notch1, as well as the Notch1 pathway downstream target genes, in Huh-7 cells. I Luciferase reporter for the Notch1 in 293T cells with SLTM overexpression or knockdown. *P < 0.05, **P < 0.01, ***P < 0.001, ns no significance.