Figure 1

ASB2 is a novel E3 ligase of SMAD9. (a) Independent databeses of interactome were used to narrow the candidates for SMAD9-binding proteins. Finally, 42 proteins were remained (see TableTable1).1). (b–d) HEK293T cells were transfected with vectors encoding indicated tagged proteins and Flag-immunoprecipitates or lysates were separated using SDS-PAGE and immunoblotted with the indicated antibodies. (b) ASB2 but not DNAJA3 ubiquitilated SMAD. (c) ASB2 interacts SMAD9. (d) The amounts of transfected vector encoding Myc-Asb2 were 0, 0.1, 1, and 2 µg, respectively (from the left column) The total amount of the transfected vector was adjusted using empty vector. The protein amount of SMAD9 was negatively correlated with that of ASB2. (e) Pulse chase analysis. To terminate further protein synthesis, cells were treated with 50 µg/mL cycloheximide with or without 10 µM lactacystin at 24 h after transfection. Total cell lysates were obtained at the indicated times and subjected to western blot analysis. Protein levels were determined using densitometry. (f) Asb2 suppress BMP signalling through degradation of Smad9. HeLa cells were co-transfected with Xvent2-Luc plasmid, which expresses luciferase under BMP signalling, and a pGL4.75 vector as a control, which constantly expresses Renilla luciferase, and vectors encoding Flag-SMAD9, Flag-SMAD4 and Myc-ASB2 as indicated. Cells were treated with 100 µM BMP2. Using cell lysates obtained 24 h after transfection, the luminescence of both firefly and Renilla luciferase were measured. Luciferase activity under BMP signalling was normalized with Renilla luciferase activity. The cropped gel images were delineated and uncropped gel images are shown in the Supplementary Information. The data are shown as the mean ± s.d. Statistical analysis was performed using one-way ANOVA with Tukey’s multiple comparison tests. *p < 0.05; ns, not significant.