Fig. 5

Subcellular localization, dimerization and DNA binding of BTas proteins. a HeLa cells (5 × 10⁴) were transfected with pcDNA3.1(+), BTas-Y, BTas-B or BTas-B-N108S. Indirect immunofluorescence was performed 48 h post-transfection to visualize subcellular localization of different BTas proteins (with rabbit anti-BTas antibody at 1:400 dilution and FITC-conjugated goat anti-rabbit secondary antibody at 1:500 dilution). Nuclei were stained with DAPI (0.2 μg/mL). b HEK293T cells were transfected with BTas-Y, BTas-B or empty vector. Forty-eight hours later, cell lysates were incubated with or without the cross-linking buffer followed by Western blotting. Positions of BTas monomers (M) and dimers (D) are indicated. c EMSAs were performed with purified GST-BTas-B, GST-BTas-Y and GST-BTas-B-N108S and GST proteins. DNA probes from BFV-LTR TRE (−368/-346) or BFV-IP TRE (9243–9264) were labeled with digitonin. d Recombinant proteins GST-BTas-B, GST-BTas-Y, GST-BTas-B-N108S and GST used in EMSA were detected by Coomassie blue staining. (e) DNA binding ability of BTas was analyzed using a mammalian expression system in HEK293T cells. HEK293T cells (2.5 × 10⁵) were transfected with 100 ng empty vector, pCMV-AD-BTas-B (1–133 aa) or pCMV-AD-BTas-Y (1–133 aa) together with 20 ng pBFV-LTR-Luc or pBFV-IP-Luc. Forty-eight hours later, luciferase activities were measured as described in Materials and Methods. f Experiments in e were carried out in Cf2Th cells. All results shown are the average from three independent experiments. Error bars indicate SD. *P < 0.05 (unpaired t test), **P < 0.01 (unpaired t test)