Fig. 2.
Processing of K48- and K63-linked polyubiquitin chains by FMDV Lbpro in vitro. (A) The protein was obtained from FMDV Lbpro-transfected or mock-transfected 293T cells using Ni Sepharose 6 Fast Flow and analyzed for His-tagged FMDV Lbpro-conjugated proteins by Western blotting (WB) with an anti-His antibody. (B) In vitro K48-linked polyubiquitin deconjugation assay. K48-linked polyubiquitin was incubated with the protein obtained from mock-transfected (lane 2) or FMDV Lbpro-transfected (lane 3) HEK293T cells at 37°C for 1 h before being analyzed by SDS-PAGE. Lane 1, uncleaved K48-linked polyubiquitin chain (K48-Ub2-7). M, molecular mass markers, including 170-, 130-, 100-, 70-, 55-, 40-, 35-, 25-, 15-, and 10-kDa bands. (C) In vitro K63-linked polyubiquitin deconjugation assay. The experiment was performed similarly to that described for panel B except that the K63-linked polyubiquitin chain (K63-Ub2-7) was used.