Figure 6.
Abi physically interacts with Notch in an Ub-dependent manner. (A and B) Analysis of the Abi-Notch interaction by coimmunoprecipitation. S2N cells were transfected with HA-Abi cDNA with or without shi dsRNA, pretreated with 0.7 mM CuSO4 for 24 h, and further incubated in the presence and absence of S2S cells for 18 h. (A) Western blots of cell lysates (input) and anti-IgG or anti-NICD immunoprecipitates, probed with an anti-HA or anti-NICD antibody. (B) Quantification of HA-Abi levels in anti-NICD immunoprecipitates by densitometry. (C) Western blots of third instar larval lysates (input) and anti-IgG or anti-NICD immunoprecipitates, probed with an anti-Abi or anti-NICD antibody. (D) Ser-induced ubiquitination of Notch. Western blots of cell lysates (input) and anti-HA immunoprecipitates from S2N cells transfected with HA-Ub cDNA, with or without shi dsRNA treatment, probed with an anti-NICD antibody. Transfected cells were incubated in the presence or absence of S2S cells and CuSO4 for 18 h prior to western blotting. White and black arrowheads mark HA-Ub–modified full-length Notch and its breakdown product, respectively. (E) Quantification of cleaved Notch levels in anti-HA immunoprecipitates by densitometry. (F and G) Direct interaction between Abi and Notch in a Ub-dependent manner. Purified recombinant GST-Abi-SH3 was incubated with His6-TF, His6-TF-NICD, His6-TF-NICD-Ub, or His6-TF-Ub. (F) Western blot of His6 pull-downs probed with an anti-GST antibody (upper panel). The lower panel shows Coomassie blue staining of His6-TF proteins. (G) Quantification of GST-Abi-SH3 levels in His6 pull-downs by densitometry. (H and I) Analysis of the AbiΔSH3-Notch interaction. Cell lysates were prepared from S2N cells transfected with HA-Abi or HA-AbiΔSH3 cDNA, with or without shi dsRNA as in A. (H) Western blots of cell lysates (input) and anti-IgG or anti-NICD immunoprecipitates, probed with an anti-HA or anti-NICD antibody. (I) Quantification of HA-Abi and HA-AbiΔSH3 levels in anti-NICD immunoprecipitates by densitometry. Data represent the mean ± SEM. n = 3 independent experiments. Statistical analyses were performed using a one-way ANOVA with the Tukey–Kramer post hoc test (*P < 0.05; **P < 0.01; ***P < 0.001). Source data are available for this figure: SourceData F6.

Abi physically interacts with Notch in an Ub-dependent manner. (A and B) Analysis of the Abi-Notch interaction by coimmunoprecipitation. S2N cells were transfected with HA-Abi cDNA with or without shi dsRNA, pretreated with 0.7 mM CuSO4 for 24 h, and further incubated in the presence and absence of S2S cells for 18 h. (A) Western blots of cell lysates (input) and anti-IgG or anti-NICD immunoprecipitates, probed with an anti-HA or anti-NICD antibody. (B) Quantification of HA-Abi levels in anti-NICD immunoprecipitates by densitometry. (C) Western blots of third instar larval lysates (input) and anti-IgG or anti-NICD immunoprecipitates, probed with an anti-Abi or anti-NICD antibody. (D) Ser-induced ubiquitination of Notch. Western blots of cell lysates (input) and anti-HA immunoprecipitates from S2N cells transfected with HA-Ub cDNA, with or without shi dsRNA treatment, probed with an anti-NICD antibody. Transfected cells were incubated in the presence or absence of S2S cells and CuSO4 for 18 h prior to western blotting. White and black arrowheads mark HA-Ub–modified full-length Notch and its breakdown product, respectively. (E) Quantification of cleaved Notch levels in anti-HA immunoprecipitates by densitometry. (F and G) Direct interaction between Abi and Notch in a Ub-dependent manner. Purified recombinant GST-Abi-SH3 was incubated with His6-TF, His6-TF-NICD, His6-TF-NICD-Ub, or His6-TF-Ub. (F) Western blot of His6 pull-downs probed with an anti-GST antibody (upper panel). The lower panel shows Coomassie blue staining of His6-TF proteins. (G) Quantification of GST-Abi-SH3 levels in His6 pull-downs by densitometry. (H and I) Analysis of the AbiΔSH3-Notch interaction. Cell lysates were prepared from S2N cells transfected with HA-Abi or HA-AbiΔSH3 cDNA, with or without shi dsRNA as in A. (H) Western blots of cell lysates (input) and anti-IgG or anti-NICD immunoprecipitates, probed with an anti-HA or anti-NICD antibody. (I) Quantification of HA-Abi and HA-AbiΔSH3 levels in anti-NICD immunoprecipitates by densitometry. Data represent the mean ± SEM. n = 3 independent experiments. Statistical analyses were performed using a one-way ANOVA with the Tukey–Kramer post hoc test (*P < 0.05; **P < 0.01; ***P < 0.001). Source data are available for this figure: SourceData F6.

This Feature Is Available To Subscribers Only

or Create an Account

Close Modal
Close Modal