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. Author manuscript; available in PMC: 2020 Feb 15.
Published in final edited form as: Virology. 2019 Oct 20;540:104–118. doi: 10.1016/j.virol.2019.10.005

Fig. 6. Validation of Rab11B, Importin and Crm-1 interactions with Agno.

Fig. 6.

(A) Analysis of GST, GST-Agno and GST-Agno mutant (L33D + E34L) fusion proteins by SDS-10% PAGE. GST and GST-Agno were produced in bacteria and affinity purified as previously described (Saribas et al., 2011). Five micrograms of each protein was loaded on the gel, resolved and stained by Coomassie blue. (B and C) Analysis of the interaction of Agno with Rab11B and Crm-1 by GST pull-down assays. Whole-cell extracts (0.5 mg) prepared from SVG-A cells transfected with either flag-tagged-Rab11B or flag-tagged Crm-1 expression plasmids (lanes 4, and 5 on each panel) were incubated with either GST alone (lane 4) or GST-Agno (lane 5). After washing, proteins interacting with GST or GST-Agno were analyzed by Western blotting using an anti-flag antibody for detection of flag-tagged Rab11B and flag-tagged Crm-1. (D) Analysis of bacterially-produced GST and GST-Crm-1 fusion protein by SDS-10% PAGE. GST-Crm-1 was produced as described previously (Dong et al., 2009). Two micrograms of GST and GST-Crm-1 were loaded on the gel, resolved and stained by Coomassie blue. (E) Interaction of Agno with Crm-1. Whole-cell extracts prepared from SVG-A cells infected with JCV Mad-1 strain (14th day post-infection) were incubated with either GST alone (lane 4) or GST-Crm-1 (lane 5). After washing, proteins interacting with GST or GST-Crm-1 were analyzed by Western blotting using an anti-Agno antibody. On panels B, C and E, whole-cell extracts prepared from untransfected or uninfected or transfected or infected cells were loaded as negative (− Cont.) and positive (+Cont.) controls as indicated. A star on panel E points to a nonspecific interaction of a host protein with GST. Transf.: Transfection. Infect.: Infection.