The Highly Conserved Codon following the Slippery Sequence Supports -1 Frameshift Efficiency at the HIV-1 Frameshift Site

Mathew, Suneeth F.; Crowe-McAuliffe, Caillan; Graves, Ryan; Cardno, Tony S.; McKinney, Cushla; Poole, Elizabeth S.; Tate, Warren P.

Total cell lysates were harvested 48 h or 120 h after transfection as described. Total protein content was determined by BCA assay [47] and verified by Coomassie-stained polyacrylamide gels. Total protein (25 μg) was separated by 12.5% (w/v) SDS-PAGE and transferred to Protran nitrocellulose membranes (Whatman Schleicher & Schuell). Membranes were stained with Ponceau S staining solution (0.1% (w/v) Ponceau S, 5% (v/v) acetic acid) for 1-2 min, rinsed with dH 2 O, and the positions of lanes and marker bands were marked with a pencil. Membranes were then blocked for 1 h at 4°C in TBST (40 MM Tris-HCl pH 7.6, 150 MM NaCl, 0.05% (v/v) Tween 20) containing 5% (w/v) milk powder, incubated overnight at 4°C with rabbit anti-eRF1 [48] (1:10000 dilution, a kind gift from Dr Adam Geballe (Fred Hutchinson Cancer Research Center, Seattle, USA)) in TBST containing 5% (w/v) BSA, washed (3× with TBST), incubated (1 h at 4°C) with rabbit anti-β-actin (Sigma, 1:5000 dilution) and 2% (w/v) milk powder, and washed (3×, with TBST). Finally, the membranes were incubated (1 h at 4°C) with goat anti-rabbit-HRP (Sigma, 1:5000 dilution) in TBST containing 5% (w/v) milk powder, and washed (3×, with TBST) before ECL Western Blotting Detection Reagents (Amersham Biosciences) were applied according to the manufacturer's instructions and the membranes exposed to X-ray film. eRF1 expression was quantified relative to β-actin and the non-transfected control using the ImageQuant™ TL software (Applied Biosystems).