Pulse Label and Chase.

HeLa cells were plated onto 6-cm tissue culture dishes (Sarstedt) and transfected with 1 μg of pCAGGS W3A F, pCAGGS S443D F, or empty pCAGGS vector (MCS) as described above. At 16 h posttransfection, the cells were washed 3× with PBS⁺ and starved for 30 min with 3 mL of DMEM lacking cysteine and methionine. After 30 min, the medium was replaced with 1 mL of DMEM (lacking cysteine and methionine) supplemented with [³⁵S]-Promix (50 μCi per dish) and returned to 37 °C/5% CO₂ for 20 min (the “pulse”). After 20 min, the radioactive DMEM was aspirated, replaced with 5 mL of “chase” media (DMEM + 10% FBS + 1% penicillin/streptomycin), and incubated at 37 °C/5% CO₂ for 0, 15, 30, 45, 60, and 90 min. The control dishes were stopped at 0 and 90 min chase. At each time point, cells were transferred to ice to minimize protease activity, washed 2× with PBS⁻, and lysed using 1 mL of 1× radioimmunoprecipitation assay (RIPA) buffer (52) supplemented with protease inhibitors, 1 mM phenylmethylsulfonyl fluoride, and 10 mM iodoacetamide. The cell lysate was clarified by ultracentrifugation at 55,000 rpm in a Beckman TLX ultracentrifuge with a TLA 120.2 rotor at 4 °C for 10 mins, transferred to a 1.5-mL Eppendorf tube, and kept on ice. Primary antibody (α-PIV5 F rabbit polyclonal R9176) was added at a 1:100 dilution, and samples were rocked for 2 h at 4 °C. After primary antibody binding, 35 μL of protein A Sepharose was added to each sample and samples were rocked for 30 min at 4 °C. The beads were pelleted using a tabletop centrifuge and washed 3× with RIPA buffer containing 0.3 M NaCl, 2× with RIPA buffer containing 0.15 M NaCl, and 1× with a 50 mM Tris⋅HCl (pH 7.4), 0.25 mM EDTA, 0.15 M NaCl solution. Proteins were eluted from beads by boiling for 3 min in protein lysis buffer containing 15% DTT and analyzed by SDS/PAGE electrophoresis on a 15% polyacrylamide gel. Radioactivity was detected using a Fuji FLA-5100 image reader with Multi Gauge v3.0 software (Fuji Medical Systems).