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. 2023 Dec 26;20(3):645–658. doi: 10.1080/15548627.2023.2299514

Figure 6.

Figure 6.

Ubiquitination of ATG4B by UBE3C inhibits ATG4B activity and ATG4B-LC3 interaction. (A) analysis of the effect of UBE3C on the activity of ATG4B. The test operation is as described in the method gel-based assay. LC3-GST was used as ATG4B substrate to measure the activity of ATG4B in cell lysate after UBE3C overexpression or knockdown. Experimental systems were analyzed by Coomassie brilliant blue staining. The rest LC3-GST were quantified. n = 3, **P < 0.01, ***P < 0.001. (B) the activity of ATG4B in ATG4B KO HeLa cells stably expressing GFP-ATG4BK119R was tested when UBE3C overexpressed. Experimental systems were analyzed by Coomassie brilliant blue staining. The rest LC3-GST were quantified. n = 3, ns: no significance. (C) 293T cells transfected with his-UBE3C for 24 h and stably expressing sh-UBE3C were incubated with EBSS for 1 h. The enzymatic activity of ATG4B were quantified. n = 3, ***P < 0.001. (D) the activity of ATG4B in ATG4B KO HeLa stably expressing GFP-ATG4B and GFP-ATG4BK119R with or without EBSS treatment for 1 h was detected. Experimental systems were analyzed by Coomassie brilliant blue staining as well. n = 3, ***P < 0.001, ns: no significance. (E) IP analysis of the interaction between flag-ATG4B and GFP-LC3 in 293T cells. 293T cells stably expressing GFP-LC3 were transfected with flag-ATG4B. In the case of UBE3C overexpression or knockdown, the GFP-LC3 were pulled down by anti-flag-gel from 293T cells transfected with indicated constructs. Western blotting with indicated antibodies and quantification of GFP-LC3:Flag-ATG4B in IP fraction was performed. n = 3, *P < 0.05. (F) 293T cells were transfected with Vector, his-UBE3C and GFP-LC3, in the case of EBSS-induced starvation for 1 h or not, the interaction between endogenous ATG4B and GFP-LC3 was analyzed by IP, the GFP-LC3 were pulled down by Magnetic Beads coupled with ATG4B antibody. Western blotting with indicated antibodies and quantification of GFP-LC3:ATG4B in IP fraction was performed. n = 3, ***P < 0.001. (G) ATG4B KO HeLa cells stably expressing GFP-ATG4B and GFP-ATG4BK119R treated with EBSS for 1 h or not, the interaction between GFP-ATG4B or GFP-ATG4BK119R and endogenous LC3 was tested. Quantification of LC3:GFP-ATG4B in IP fraction was performed. n = 3, **P < 0.01, ns: no significance.