Confirmation of the interaction between SARS-CoV S protein and eIF3f by co-immunoprecipitation and immunofluorescence.

<p>a. Interaction of eIF3f with the N-terminal portion of SARS-CoV S protein (SΔC). HeLa cells expressing the Myc-tagged SΔC (lane 1), the Flag-tagged eIF3f (lane 2) and eIF3f+SΔC (lane 3) were harvested at 24 hours post-transfection and lysed. The total lysates were either detected directly by Western blot with anti-Myc (top panel) and anti-Flag (second panel) antibodies, respectively, or immunoprecipitated with anti-Flag antibody. The precipitates were analyzed by Western blot with anti-Flag (third panel) and anti-Myc (bottom panel) antibodies. b. Interaction of eIF3f with the full-length SARS-CoV S protein. HeLa cells expressing the SARS-CoV S (lane 1), the Flag-tagged eIF3f (lane 2) and eIF3f+S (lane 3), were lysed. The total lysates were either detected directly by Western blot with anti-S (top panel) and anti-Flag (second panel) antibodies, respectively, or immunoprecipitated with anti-S antibodies. The precipitates were analyzed by Western blot with anti-S (third panel) and anti-Flag (bottom panel) antibodies. c. Subcellular localization of eIF3f and SARS-CoV S protein by immunofluorescent staining of HeLa cells expressing eIF3f (panel A), SARS-CoV S protein (panel B) or co-expressing eIF3f and S (panels C, D and E). The subcellular localization of the proteins were examined at 12 hours post-transfection by dual labelling with a mixture of anti-S (rabbit) and anti-Flag (mouse) antibodies, followed by incubating with a mixture of FITC-conjugated anti-mouse and tetramethyl rhodamine isocyanate-conjugated anti-rabbit antisera. Panel C shows the staining profile of eIF3f, panel D shows the staining pattern of SARS-CoV S protein, and panels E shows the overlapping images C and D.