(A) Diagrams of mutant TRIM5γ constructs. (B) 293T cells were co-transfected with FLAG-HBx and TRIM5γ-derived constructs or EV as indicated, and whole-cell lysates were immunoblotted with anti-FLAG, HA, or GAPDH antibodies. (C) 293T cells were transfected as indicated; 28 h later, coIP was carried out, and cell lysates and precipitated samples were analyzed by immunoblotting using an anti-FLAG or anti-HA antibody. (D) 293T cells were transfected with glutathione S-transferase (GST)-BBox, flag-HBx, or EV as indicated and were subjected to coIP analysis as in (C). (E) 293T cells were co-transfected with FLAG-HBx and GST-BBox or HA-TRIM5γ plasmids and analyzed as in (B). (F) HepG2 cells were transfected with FLAG-HBx, HA-Ub, GST-Bbox, or GST-EV plasmids as indicated; 24 h later, cells were treated with MG132 for 8 h, collected, and subjected to coIP analysis. Data are representative of at least three independent experiments.