Fig. 2: MAML1 acts as a negative regulator of Itch leading to K63-linked Itch self-ubiquitylation. | Cell Death & Differentiation

Fig. 2: MAML1 acts as a negative regulator of Itch leading to K63-linked Itch self-ubiquitylation.

From: MAML1 drives Notch and Hedgehog oncogenic pathways by inhibiting Itch activity in triple-negative breast cancer

Fig. 2

A Representative immunoblotting of Itch, Gli1, Notch1, and MAML1 from lysate of NIH3T3 and Ptch–/– MEFs upon small interference of MAML1 (siMAML1) or control cells (siScr). Lower panel, densitometric analysis of Itch protein levels: NIH3T3 7.4-fold increase; Ptch–/– MEFs 4.3-fold increase. B, C In vitro ubiquitination assay, Myc-tagged Itch was immunoprecipitated with α-Myc antibody from lysates of WT MEFs co-transfected with indicated plasmids described in the panels, or in a high percentage of SDS (1%) (C), followed by immunoblotting with α-HA antibody to detect the Itch polyubiquitylated forms. α-Myc antibody was used to re-probe blots to assess the levels of immunoprecipitated protein. The lower panels show the immunoblots of Pre-IP WCEs by using α-Flag antibody. D In vitro ubiquitination assay of endogenous Itch from lysates of WT MEFs, transfected as described in the figure. Immunoprecipitation of Itch with α-Itch antibody and immunoblotting for α-HA antibody to detect the Itch polyubiquitylated forms. α-Itch antibody was used to re-probe blots to assess the levels of immunoprecipitated protein. The lower panels show the immunoblots of Pre-IP WCEs by using α-Flag antibody. E MAML1 induces Itch self-ubiquitination in WT MEFs transfected with indicated plasmids described in the panel. The cell lysates were immunoprecipitated with α-Flag antibody, immunoblotted for α-HA to detect the Itch polyubiquitylated forms. α-Flag antibody was used to re-probe blots to assess the levels of immunoprecipitated protein. The lower panels show the immunoblots of Pre-IP WCEs by using α-MAML1 antibody. F In vitro ubiquitination assay of Myc-Itch from lysates of WT MEFs transfected as indicated. Proteins were immunoprecipitated with α-Myc antibody and immunoblotted with α-HA antibody to detect the Itch polyubiquitylated forms. α-Flag antibody was used to re-probe blots to assess the levels of immunoprecipitated protein. The lower panels show the immunoblots of Pre-IP WCEs by using α-Flag antibody. Densitometric analysis in panel A normalized on endogenous Tubulin; data represent Mean ± SEM of n = 3 (NIH3T3) and n = 6 (Ptch–/– MEF) independent experiments; *p < 0.05, **p < 0.01 calculated by two-tailed ratio paired t test. Representative immunoblotting of at least n = 3 biological replicates with similar results are shown in B–F. Tubulin was used as the loading control. The arrows indicate the molecular weight of the immunoprecipitated protein.

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