TRIM21 restricts HBV DNA replication. (A) Huh7 cells were cotransfected with pHBV1.3 and pcDNA3 or TRIM21 plasmid or pSilencer-NC or shR-TRIM21. qPCR was used to detect HBV DNA. (B) HepG2.2.15 cells were transfected with pcDNA3, TRIM21, pSilencer-NC or shR-TRIM21. HBV DNA was detected as described in (A). (C) HepG2.2.15 cells were transfected with pcDNA3 or TRIM21, and Southern blot was used to detect the cellular replicative intermediate DNA (RI-DNA). (D) Huh7 cells were transfected with pHBV1.3, pcDNA3 (left panel) or pTRIM21 (right panel) and ubiquitin or K48R or K63R. HBV DNA was detected by qPCR. (E) Except for pHBV1.3, HepG2.2.15 cells were transfected as described in (D), and the relative levels of HBV DNA were detected in HepG2.2.15 cells. (F) Huh7 cells were cotransfected with pHBV1.3 and pcDNA3 or TRIM21-ΔSPRY, and the HBV DNA level was detected by qPCR. (G) Except for pHBV1.3, HepG2.2.15 cells were transfected as described in (F). (H) HepG2.2.15 cells were transfected with pcDNA3 or TRIM21-ΔSPRY, and RI-DNA was examined by Southern blot. Data are representative of three independent experiments with three replicates each. The data represent the means ± S.D. * P < 0.05, ** P < 0.01, *** P < 0.001.