Cells were grown at 37 °C in a humidified atmosphere with 5% CO2 in DMEM (HeLa, 293-ampho) or 1:1 DMEM:F12 media (RPE1) from Invitrogen supplemented with 10% fetal bovine serum (Atlanta Biologicals), 1X penicillin-streptomcyin and non-essential amino acids (Invitrogen), and 2 mM L-glutamine (Invitrogen). Cells used for live imaging or immunofluorescence were grown on no. 1.5 glass coverslips (Fisher Scientific) coated with Poly-D-lysine (Sigma). 293-ampho cells were transfected according to a calcium phosphate protocol to generate retroviruses. Virus-containing media was supplemented with 4 µg/mL polybrene (Sigma) and applied to target cells, followed by selection with puromycin (Sigma). Nocodazole (Sigma), MG132 (Boston Biochem), ZM447439 (Tocris Bioscience), BI2536 (Selleck Biochemicals) and Hesperadin (synthesized in the Kapoor lab) were dissolved in DMSO. For siRNA treatments, 1.7 × 105 RPE1 cells were transfected with 150 pmol siRNA and 7.5 µL Lipofectamine RNAi Max (Invitrogen) following the manufacturer’s reverse transfection protocol and immediately plated onto no 1.5 glass coverslips. In all experiments, cells were fixed or lysed 42–46 h post-transfection (Fisher Scientific). An siRNA targeting mCherry (5’-GCUCCAAGGCCUACGUGAAUU-3’) was used for control transfections.
To generate B56 family siRNA pools, two individual siRNAs from Dharmacon smart pools for each B56 gene determined to be effective in reducing protein levels of endogenous and/or GFP-tagged B56 genes were chosen. siRNAs were mixed at equimolar ratios, with the exception of B56ε siRNA, which was included at 1.5-fold relative to the other siRNAs. siRNAs used in the B56 family pools are: B56α: GCUCAAAGAUGCCACUUCA (pool 1), and UGAAUGAACUGGUUGAGUA (pool 2); B56β: CGCAUGAUCUCAGUGAAUA (pool 1), and GAACAAUGAGUAUAUCCUA (pool 2); B56γ: GGAUUUGCCUUACCACUAA (pool 1), and GGAAGAUGAACCAACGUUA (pool 2); B56δ: UCCAUGGACUGAUCUAUAA (pool 1), and UGACUGAGCCGGUAAUUGU (pool 2); B56ε: UUAAUGAACUGGUGGACUA (pool 1), and GCACAGCUGGCAUAUUGUA (pool 2).
To generate B56 family siRNA pools, two individual siRNAs from Dharmacon smart pools for each B56 gene determined to be effective in reducing protein levels of endogenous and/or GFP-tagged B56 genes were chosen. siRNAs were mixed at equimolar ratios, with the exception of B56ε siRNA, which was included at 1.5-fold relative to the other siRNAs. siRNAs used in the B56 family pools are: B56α: GCUCAAAGAUGCCACUUCA (pool 1), and UGAAUGAACUGGUUGAGUA (pool 2); B56β: CGCAUGAUCUCAGUGAAUA (pool 1), and GAACAAUGAGUAUAUCCUA (pool 2); B56γ: GGAUUUGCCUUACCACUAA (pool 1), and GGAAGAUGAACCAACGUUA (pool 2); B56δ: UCCAUGGACUGAUCUAUAA (pool 1), and UGACUGAGCCGGUAAUUGU (pool 2); B56ε: UUAAUGAACUGGUGGACUA (pool 1), and GCACAGCUGGCAUAUUGUA (pool 2).
