FIG 1.

Vimentin is identified as a DENV NS4A-binding protein. (A) Huh-7 cells stably transfected with the pcTAP-NS4A or pcTAP vector (control) constructs were lysed, and the DENV-2 NS4A protein complexes were sequentially purified first by using streptavidin resin and then by using calmodulin resin. The samples were resolved on a 10% SDS-polyacrylamide gel, and bands were visualized using Coomassie blue staining. The box in the NS4A-TAP lane indicates the band identified as vimentin by mass spectrometry, and the black arrows mark the positions of the seven other proteins identified. The positions of the protein markers (M) (in kilodaltons) are indicated to the left of the gel. (B) Huh-7 cells were infected with DENV-2 at an MOI of 10 and lysed at the three stipulated time points. Infected and mock-infected total cell lysates were incubated with vimentin, NS4A, or IgG isotype antibodies and Dynabeads protein G were added to capture the protein complexes. Samples with eluted target proteins were analyzed by Western blotting using anti-NS4A or antivimentin antibodies. Mock-infected samples and IgG isotype control are shown in gels 1 and 2, respectively, and are the negative controls. Gels 3 and 4 show the infected samples in which Co-IP of NS4A with vimentin and its reciprocal assay, respectively, confirmed the interaction. The specific protein bands representing vimentin and NS4A are shown by arrows to the right of the gels.