ON-TARGETplus siRNA oligos (Dharmacon) were used for RNAi-mediated KD experiments. Scrambled sequence for control siRNAs: (D-001810-01) 5′-UGGUUUACAUGUCGACUAA-3′; (D-001810-02) 5′-UGGUUUACAUGUUGUGUGA-3′; (D-001810-03) 5′-UGGUUUACAUGUUUUCUGA-3′; (D-001810-04) 5′-UGGUUUACAUGUUUUCCUA-3′. NUP153 siRNAs: (J-057025-11) 5′-CGCUAUGUGCAUUGAUAAA-3′; (J-057025-12) 5′-GGGACAGGCUUUGGAGAUA-3′. ELYS siRNA: (J-051465-09) 5′-CCACUGAACUAACUACUAA-3′; (J-051465-10) 5′-GGAAAGAAGAAGGACGUUA-3′. TPR siRNA: (J-041152-09) 5′-CAACAAACAUUCAUCGGUA-3′; (J-041152-10) 5′-CGUGACAUGUACCGAAUUU-3′.
5 × 104 MEFs were plated into each well of 6-well plates 24 h before transfection. 30 pmol of siRNA oligos was transfected onto the cells in each well with Lipofectamine RNAiMAX transfection reagents (Thermo Fisher Scientific), following the manufacturer’s instructions. 48 h after incubation at 37°C, the transfected cells were trypsinized and replated at 5 × 104 cells/well into each well of 6-well plates and transfected with 30 pmol of the siRNA. 48 h after incubation at 37°C, the transfected cells were trypsinized and replated on coverslips for indirect immunofluorescence or plated into a 60-mm dish for Western blotting.
5 × 104 MEFs were plated into each well of 6-well plates 24 h before transfection. 30 pmol of siRNA oligos was transfected onto the cells in each well with Lipofectamine RNAiMAX transfection reagents (Thermo Fisher Scientific), following the manufacturer’s instructions. 48 h after incubation at 37°C, the transfected cells were trypsinized and replated at 5 × 104 cells/well into each well of 6-well plates and transfected with 30 pmol of the siRNA. 48 h after incubation at 37°C, the transfected cells were trypsinized and replated on coverslips for indirect immunofluorescence or plated into a 60-mm dish for Western blotting.