Figure 4.

RNF31 and OTULIN balance ATG13 protein level and the autophagic flux. (A) MYC-ATG13 ubiquitination under different conditions. HEK293 FT cells were transfected with MYC-ATG13 and untagged ubiquitin, along with the control, or RNF31 siRNA-1, or OTULIN shRNA-1, or (OTULIN shRNA-1 + RNF31 siRNA-1), or (RNF31 siRNA-1 + Flag-OTULIN), or (OTULIN shRNA-1 + Flag-RNF31) and treated with DMEM or EBSS for 4 h. Ubiquitinated proteins were immunoprecipitated with anti-MYC beads under denaturing conditions and immunoblotted with an anti-ubiquitin antibody. (B) LC3B puncta formation was analyzed in HeLa cells stably expressing mRFP-EGFP-LC3B. Cell were transfected with the control, or RNF31 shRNA-1, or OTULIN shRNA-1, or (OTULIN shRNA-1 + RNF31 shRNA-1), or (OTULIN shRNA-1 + Flag-RNF31), or (RNF31 shRNA-1 + Flag-OTULIN). Cells were treated with EBSS for 4 h. Cells were fixed and stained with DAPI (blue) and representative fluorescence images are shown. Scale bar: 5 μm. (C) Statistical analysis of the number of yellow (RFP⁺GFP⁺) and red (RFP⁺GFP⁻) puncta per cell as represented in (B) and Figure S4. Data are mean ± SD from three independent experiments. *p < 0.05; **p < 0.01; ***p < 0.001 (one-way ANOVA). (D) Western blot analysis of the samples as represented in (B) and Figure S4. The knockdown efficiency and protein overexpression were shown. Samples were also immunoblotted with the indicated antibodies. The numbers are the relative ratio of LC3B-II/I