Fig. 4

TRIM21 decreases the expression of lipogenic enzymes by mediating ubiquitination-mediated degradation of SREBF1. TRIM21 knockdown (A) and overexpression in ACHN (B) can increase and decrease metabolic enzymes above the transcriptional level, with the exception of ACLY and SREBF. TRIM21 knockdown (C) and overexpression in ACHN (D) can increase and decrease metabolic enzymes above at the protein level except ACLY. ACHN was transfected with exogenous HA-TRIM21 and Flag-SREBF1. Anti-FLAG (E) and anti-HA (F) antibodies were used for immunoprecipitation to detect the mutual binding between TRIM21 and SREBF1. G-H Western blot showing SREBF1 protein decay at the indicated time points after cycloheximide (50 μg/ml) addition to ACHN. I Western blot showing the effects of the proteasome inhibitor MG132 (10 μM for 6 h) treatment on SREBF1 protein accumulation. J TRIM21 was overexpressed in ACHN cells, and immunoprecipitation was performed using an Anti-SREBF1 antibody, which suggested that TRIM21 could bind to SREBF1 and increase the ubiquitination degradation level of SREBF1. K HEK293T cells were transfected with TRIM21 WT or the indicated mutant for 48 h harvested and subjected to IP with anti-HA beads. L K48-only and K63-only ubiquitin-HA plasmids alone or co-transfected with TRIM21 and Flag-SREBF1 plasmid into HEK293T cells for 48 h. Then, the cells were harvested and subjected to IP with anti-Flag beads. All the results were confirmed by three times repeated experiments. Statistical analysis was performed using unpaired t-tests. All statistical tests were two-sided. *p < 0.05, **p < 0.01, ***p < 0.001, ns, no significance