*2.5. Metabolic Labeling*

For pulse-chase analysis, MDCK-2 cells transiently expressing CedV F proteins were incubated at 24 h p.t. for 15 min with medium lacking cysteine and methionine, followed by incubation with medium containing [35S]cysteine and -methionine (Hartmann Analytic, Braunschweig, Germany) at a final concentration of 100 μCi/mL for 10 min (pulse). Then, labeling medium was replaced by a serum-free nonradioactive medium, and cells were incubated at 37 ◦C for 2 h (chase). Afterwards, cells were washed with PBS, followed by cell lysis in radioimmunoprecipitation assay (RIPA) buffer (1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS, 0.15 M NaCl, 10 mM EDTA, 50 units/mL aprotinin, and 20 mM Tris-HCl, pH 7.5). Cell lysates were centrifuged for 45 min at 20,000× *g*, and CedV F proteins were immunoprecipitated using a polyclonal anti-HA antibody (H6908; 1:500; Sigma, Darmstadt, Germany). Protein A-Sepharose CL-4B (Sigma, Darmstadt, Germany) suspension was added for another 45 min followed by several washes of the immune complexes with RIPA buffer. After suspension in sample buffer, proteins were separated on a 12% polyacrylamide gel under reducing conditions. Dried gels were subjected to autoradiography and analyzed with a CR35 Dark Box Image analyzer (Duerr Medical, Bietigheim-Bissingen, Germany).
