Knockdown of Vps26 and Vps35 in Cell Lines

Adherent cells were seeded at a density of 3×10⁵ per well and transfected with Dharmacon Smartpool targeting human Vps26A, Vps35 or a non-targeting control siRNA using Oligofectamine based on the method of Seaman [18] (link). Unless otherwise stated, 200 nM of siRNA was used. Twenty-four hours later the cells were reseeded and the following day an identical second transfection was performed. Forty-eight hours later the cells were harvested and Vps26 and Vps35 knockdown were determined by western blotting. HeLa TZM-bl cells were infected with pNL4.3 or pNL4.3-Δ144 virus at an MOI of 0.1 immediately after the second knockdown. Excess virus was removed by washing and cells incubated at 37°C for 48 h prior to analysis. RNAi knockdown of Vps26 in T cells was performed by generating 4 individual oligonucleotide hairpins based on the Dharmacon Smartpool target sequences. shRNA hairpins were cloned individually into the HIV-1 vector pCSRQ, a modified version of pSIREN RetroQ [59] (link) (a gift from G. Towers, UCL) and co-transfected into 293T cells with the VSV-G envelope plasmid. Virions were used to infect T cells and stable cell lines selected with puromycin.