Journal: Proceedings of the National Academy of Sciences of the United States of America
Article Title: Influenza virus targets the mRNA export machinery and the nuclear pore complex
Figure Lengend Snippet: The NS1 protein of influenza virus interacts with key constituents of the mRNA export pathway. ( a ) Cell lysates from 293T cells were incubated with immobilized recombinant GST or GST-NS1. Bound and unbound (UB) fractions were analyzed by 4–20% SDS/PAGE followed by immunoblot analysis with antibodies to NXF1, p15, Rae1, E1B-AP5, and Nup98. ( b and c ) Experiments were performed as in a except that antibodies against Nup96 and against Nup62 and Nup153 (mAb414) were used for immunoblot analysis. ( d ) GST-NS1 or the amino-terminal or carboxyl-terminal domains of NS1 fused with GST were incubated with cell lysates and processed as in a . ( e ) GST-NS1 was incubated with cell lysates untreated or treated with RNase A and processed as in a . ( f and g ) Expression levels of Nups and mRNA export factors in 293T ( f ) and MDCK ( g ) cells infected with influenza virus. Cell extracts were subjected to immunoblot analysis with antibodies against Nup98, β-actin, Rae1, NXF1, and E1B-AP5, and with mAb414 antibodies. ( h ) Half-life measurements of Nup98. MDCK cells were pulse-labeled for 2 h and chased for the depicted time points. Immunoprecipitations were performed with anti-Nup98 antibodies or preimmune serum (PI). Nup98 bands were analyzed by densitometry as described in Methods .
Article Snippet: Immunoblot analysis was performed with anti-Nup98 , anti-E1B-AP5 (Proteintech Group, Chicago, IL), anti-Rae1 , and anti-NXF1/TAP antibodies (BD Transduction Laboratories, San Jose, CA), anti-p15/NXT antibodies (Abnova, Taipei City, Taiwan), mAb414 , and anti-Influenza A antibodies (virions; Biodesign International, Saco, ME).
Techniques: Incubation, Recombinant, SDS Page, Expressing, Infection, Labeling