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. 2022 Aug 8;12(13):5949–5970. doi: 10.7150/thno.72826

Figure 4.

Figure 4

The E3 ligase Cdh1 interacts with and downregulates MAST1 protein. (A) HeLa cells were transfected with a panel of E3 ligases, and the expression of MAST1 protein was analyzed using Western blotting. (B) Interactions between endogenous and (C) exogenous Cdh1 and MAST1 proteins were analyzed in HeLa cells and HEK293 cells, respectively. Cell lysates were immunoprecipitated and immunoblotted with the indicated antibodies. Protein expression was checked using Western blotting. GAPDH was used as a loading control. (D) HeLa cells were subjected to the Duolink PLA assay to analyze the interaction between Cdh1 and MAST1 using specific antibodies. Scale bar: 10 µm. (E) Schematic representation of full length Cdh1 (1-496 aa) encoding WD40 domain (represented as Cdh1-WT), N-terminus Cdh1 (1-155 aa) lacking WD40 domain (represented as Cdh1-CTM1), and C-terminus Cdh1 (156-496 aa) encoding WD40 domain (represented as Cdh1-CTM2). Interactions between full length MAST1 and Cdh1 truncated mutants by co-immunoprecipitation and immunoblotting with the indicated antibodies (lower panel). (F) Schematic representation of full length MAST1 (1-1570 aa) encoding serine/threonine (S/T) kinase domain and PDZ domain (represented as MAST1-WT), N-terminus MAST1 (1-832 aa) encoding S/T kinase domain (represented as MAST1-MTM1), C-terminus MAST1 (833-1570 aa) encoding PDZ domain (represented as MAST1-MTM2), and C-terminus MAST1 (1118-1465 aa) lacking PDZ domain (represented as MAST1-MTM3). Interactions between full length Cdh1 and MAST1 truncated mutants by co-immunoprecipitation and immunoblotting with the indicated antibodies (lower panel). (G) The effect of Cdh1 on endogenous MAST1 protein was analyzed in HeLa cells transfected with increasing concentrations of Flag-Cdh1. (H) HeLa cells were transfected with sgRNA1 and sgRNA2 targeting Cdh1 to assess the endogenous protein levels of Cdh1 and MAST1 by Western blotting. (I) The Cdh1-mediated degradation of endogenous MAST1 protein was rescued in cells transfected with sgRNA targeting Cdh1. (J) The ubiquitination of endogenous MAST1 was analyzed by transfecting HeLa cells with Flag-Cdh1 or sgRNA targeting Cdh1 followed by immunoprecipitation with an anti-MAST1 antibody and immunoblotting with an anti-ubiquitin antibody. Protein expression was checked by Western blotting with the indicated antibodies. GAPDH was used as a loading control.