FIG. 5.
Analysis of A238L mutants in cells. (A) Transient transfection analysis of A238L wild-type and mutant proteins to detect CaN coprecipitation. Vero cells were infected with MVA-T7 and transfected with pT7-SV5-A238L (lane 1), pT7-SV5-W13 (lane 2), pT7-SV5-W20 (lane 3), pT7-SV5-B19 (lane 4), pT7-SV5-Thr207 (lane 5), or pT7-SV5-IκB (lane 6). Bovine CaN was run as a control (lane 7). Radiolabeled cell extracts were immunoprecipitated with monoclonal anti-SV5 PK tag, and immune complexes were separated by SDS-PAGE. Immunoprecipitated SV5 PK-tagged proteins were detected by autoradiography. Coprecipitation of CaN was detected by protein immunoblot analysis of immune complexes with monoclonal anti-CaN(B). wt, wild type. (B) Vero cells were infected with wild-type ASFV (Ba71V; lanes 1 and 7), recombinant ASFV expressing SV5-tagged wild-type A238L (lanes 2 and 8), or SV5-tagged mutant W13 (lanes 3 and 9), W20 (lanes 4 and 10), B19 (lanes 5 and 11), or Thr207 (lanes 6 and 12). After 3.5 h, cells were pulse-labeled with [35S]Cys/Met for 1 h and harvested either immediately (lanes 1 to 6) or after a 1-h chase period with nonradioactive medium (lanes 7 to 12). Radiolabeled cell extracts were immunoprecipitated (IP) with monoclonal anti-SV5 PK tag antibody, and immune complexes were separated by SDS-PAGE. Radiolabeled immunoprecipitated SV5 PK-tagged A238L proteins were detected by autoradiography (top). Coprecipitation of CaN was detected by protein immunoblot analysis of immune complexes with monoclonal anti-CaN(B). Samples of cell extracts prior to immunoprecipitation were analyzed by SDS-PAGE and Western blot analysis with monoclonal anti-ASFV P30 antibody or monoclonal anti-SV5 PK tag antibody to detect total cellular A238L protein (bottom).