The expression of P protein from every plasmid construct was verified by SDS–polyacrylamide gel electrophoresis (PAGE) and Western blot. 293T or BSR-T7 cells were plated in a 12-well plate at 2 × 105 cells per well in DMEM supplemented with 10% of FBS. The next day, cells were transfected with 2 μg of plasmid with jetPRIME (Polyplus). For the assessment of L stability, some cells were also treated 6 hours after transfection with 2 μM 17-DMAG. Twenty-four hours after transfection, cells were trypsinized, washed with phosphate-buffered saline (PBS), and lysed for 30 min on ice. Cells expressing P only were lysed with a urea-free lysis buffer [50 mM tris-HCl (pH 7.5), 100 mM NaCl, 1 mM EDTA, 0.1% NP-40, 5% glycerol, 1 mM dithiothreitol, and cOmplete protease inhibitor (Roche)], and cells expressing L were lysed with the same buffer supplemented with 8 M urea. Proteins were separated by SDS-PAGE and electrotransferred onto polyvinylidene difluoride membranes (Hybond-P, Amersham Biosciences). After saturation for 1 hour in tris-buffered saline (TBS) containing 0.05% Tween 20 (TBS-Tween) and 5% fat-free milk, the membranes were incubated for 2 hours with primary antibodies specific for P [either the mouse monoclonal antibody 49.21 (56) or a rabbit polyclonal antibody (37, 56) diluted 1:3000 and 1:10,000 in TBS–Tween–5% milk, respectively] or Flag/L (1:2000; mouse anti-Flag monoclonal antibody, Sigma), washed with TBS-Tween, and incubated with peroxidase-conjugated sheep anti-mouse or anti-rabbit immunoglobulin G (IgG) antibody (Promega) for 1 hour at room temperature. The membranes were then washed and incubated in Covalight reagent (Covalab), and chemiluminescent signals were measured with the VersaDoc Imaging System (Bio-Rad). All P variants migrated at their expected molecular weight. Expression levels of the P MD variants that were tested in the minigenome assay were assessed using a semiquantitative dot blot assay as described previously (73) using the two anti-P antibodies described above.

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