TRIM21 targets ATG5 directly and regulates ATG5 ubiquitination. (A) TRIM21 immunoprecipitated endogenous ATG5. ARP-1, MM.1S, MM1R, and RPMI8226 cell extracts were immunoprecipitated with an antibody against ATG5, along with the immunoglobulin G (IgG) control, followed by western blotting with an ATG5 antibody. (B) ATG5 immunoprecipitated endogenous TRIM21. ARP-1, MM.1S, and MM1R cells were lysed and immunoprecipitated with an ATG5 antibody, followed by incubation with TRIM21. (C) The GST-pull down test showed that ATG5 was directly bound to TRIM21. The GST-ATG5 fusion protein was ∼58 kDa in size. The HIS-TRIM21 protein was ∼52 kDa in size. We detected HIS-TRIM21 in the supernatant of bacterial lysates and in the supernatant of GST-ATG5 beads. GST alone was used as a negative control. GST-ATG5 and HIS-TRIM21 were purified from Escherichia coli. (D) Immunofluorescence staining assay showing TRIM21 and ATG5 subcellular colocalization in 293T cells. The nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI). Original magnification: 200×, scale bar: 100 μm. (E) TRIM21 OE 293T cells were treated with MG-132 (25 μm; 6 hours) and the whole-cell lysates (WCLs) were then collected for the detection of FLAG-TRIM21, HA-ATG5, and GAPDH protein levels by immunoblotting. The WCLs were subjected to IP using an anti-ATG5 antibody, and ubiquitinated ATG5 was evaluated with a related ubiquitin antibody.