Fyn peptide by PCR-directed mutagenesis, and the resultant construct was termed pFyn SS -M1. In addition, two membrane targeting green fluorescent protein (GFP) expression plasmids, pFyn-GFP and pLck-GFP, were generated following the same methodology as was used in the production of pFyn-M1 or pLck-M1. For the construction of bimolecular fluorescence complementation (BiFC) plasmids, sequences encoding either amino-terminal fragments (residues 1 to 173, VN) or carboxyl-terminal fragments (residues 155 to 238, VC) of the Venus fluorescence protein were fused to both the N and C termini of M1 via a protein linker (YPYDVPDYAASDIAAA) ( 22 ), which also included an HA tag sequence. The BiFC plasmids were termed M1-VN, VN-M1, M1-VC, and VC-M1, respectively. The use of a flexible linker facilitates complementation of the split fragments when they are brought into close proximity by the interacting proteins. Detailed protocols, including primers and sequence information for each construct, are available upon request. All plasmids were isolated using a Qiagen Midiprep kit (Valencia, CA), and the specific mutations were confirmed by DNA sequencing. Immunofluorescence assay (IFA) and confocal microscopy. MDCK cells infected with influenza H1N1 A/WSN/33 virus or COS-1 cells transfected with the plasmid DNAs as indicated above were fixed with 2% (wt/vol) paraformaldehyde (Electron Microscopy Sciences, Hatfield, PA) followed by permeabilization with 0.2% (vol/vol) Triton X-100. Cells were further incubated with the appropriate primary antibody directly conjugated with FITC or with the primary antibody followed by secondary antibody conjugated to FITC and were then washed briefly in phosphate-buffered saline (PBS) before being