Rat anti mlv p30 r187

0.5 × 10 6 293T cells (Invitrogen) were seeded in 6-well culture plates and co-transfected with viral DNA construct (5 μg) and either pBJ5-mSERINC3-HA or pBJ5-mSERINC5-HA or empty vector (100 ng). Cells and culture media were harvested 48 h after transfection. Cells lysates were prepared as previously described (6) . Briefly, cells were lysed in DM lysis buffer (0.5% (w/v) n-Decyl-β-D-maltopyranoside in 20 mM Tris-HCl, pH 7.5, 10 % (v/v) glycerol, 1× protease inhibitors and 25 U/ml Benzonase), centrifuged at 14,000 rpm followed by the collection of the supernatant fraction. Culture media were pelleted through a 30% sucrose cushion as previously described (11) . Cell lysates or virus pellets were mixed with 1× sample loading buffer and incubated at 37 ˚C for 15 min before resolving them on 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gels. Blots were probed using the following antibodies: Polyclonal goat anti-MLV (11) (NCI repository), rat anti-MLV p30 R187 (ATCC® CRL-1912™), rabbit anti-HA (Cell Signaling Technology), monoclonal anti-β-actin (Sigma-Aldrich), HRP-conjugated anti-rabbit (Cell Signaling Technology), -anti-rat (Cell Signaling Technology), -anti-mouse (EMD Millipore) and -anti-goat (Sigma Aldrich) were used for detection using the enhanced chemiluminescence detection kits Clarity and Clarity Max ECL (Bio-Rad).