Designing the siRNA library screen.

The screen was carried out on four sets of standard plates and four sets of plates that were “sensitised” by including siRNA targeting the AP-2 μ2 subunit in every well. The same positive and negative control siRNAs were added to the first two columns of every plate. Cells were added to the 8 sets of plates on Day 1; then on Day 2, half of the plates (2 standard and 2 sensitised) were treated with 4-HT and the other half with ethanol as a control. On Day 3, the plates were shifted to a lower temperature to enhance Nef activity, and on Day 4 the assays were carried out.